U.S. patent application number 10/238911 was filed with the patent office on 2003-09-11 for 3-epi compounds of vitamin d3 and uses thereof.
This patent application is currently assigned to Women and Infants Hospital, Women and Infants Hospital. Invention is credited to Reddy, Satyanarayana G., Uskokovic, Milan.
Application Number | 20030171605 10/238911 |
Document ID | / |
Family ID | 21944580 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030171605 |
Kind Code |
A1 |
Reddy, Satyanarayana G. ; et
al. |
September 11, 2003 |
3-Epi compounds of vitamin D3 and uses thereof
Abstract
Novel 3-epi vitamin D.sub.3 compounds having the orientation of
the substituent attached to the carbon at position 3 of the A-ring
of vitamin D.sub.3 inverted from a beta (.beta.) to an alpha
(.alpha.) configuration are described. These 3-epi vitamin D.sub.3
compounds were first identified as metabolites produced via a novel
tissue-specific metabolic pathway which catalyzes the
3-.beta.-hydroxy epimerization of vitamin D.sub.3 compounds.
Isolated 3-epimer forms of vitamin D.sub.3 compounds have been
characterized and shown to have improved biological properties
compared to their isomeric counterparts, such as reduced
hypercalcemic activity and enhanced stability in vivo. The vitamin
D.sub.3 compounds of the present invention can be used as
substitutes for natural and synthetic vitamin D.sub.3
compounds.
Inventors: |
Reddy, Satyanarayana G.;
(Barrington, RI) ; Uskokovic, Milan; (Upper
Montclair, NJ) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Women and Infants Hospital
101 Dudley Street
Providence
RI
02905
|
Family ID: |
21944580 |
Appl. No.: |
10/238911 |
Filed: |
September 9, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10238911 |
Sep 9, 2002 |
|
|
|
09080026 |
May 15, 1998 |
|
|
|
60046643 |
May 16, 1997 |
|
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Current U.S.
Class: |
552/653 |
Current CPC
Class: |
C07C 401/00
20130101 |
Class at
Publication: |
552/653 ;
514/167 |
International
Class: |
A61K 031/59; C07C
401/00 |
Claims
What is claimed is:
1. An isolated 3-epi vitamin D.sub.3 compound having the formula
(I) as follows: 24wherein the orientation of the OH groups on the
A-ring is in an .alpha.-configuration; A and C are each
independently a single or a double bond; B is a single, a double,
or a triple bond; R.sub.1 and R.sub.2 are each independently a
hydrogen or a lower alkyl; R.sub.3 and R.sub.4 are each
independently a hydrogen, a lower alkyl, a hydroxyalkyl or a
haloalkyl; X is a hydrogen or a hydroxy; and Y is a hydrogen, a
hydroxy, or an oxo group, provided that the compound is not
1.alpha.,25(OH).sub.2-3-epi-D.sub.3.
2. The compound of claim 1, wherein A is a double bond and B is a
triple bond.
3. The compound of claim 1, wherein the lower alkyl of R.sub.3 and
R.sub.4 is a C.sub.1-C.sub.4 alkyl.
4. The compound of claim 1, which is selected from the group
consisting of 1,25-dihydroxy-3-epi-16-ene-vitamin D.sub.3,
1,25-dihydroxy-3-epi-23-yne-- vitamin D.sub.3,
1,25-dihydroxy-3-epi-16-ene-23-yne-vitamin D.sub.3,
1,25-dihydroxy-3-epi-24-oxo-16-ene vitamin D.sub.3,
1,24,25-trihydroxy-3-epi-16-ene vitamin D.sub.3,
1,25-dihydroxy-3-epi-20-- epi-vitamin D.sub.3, and
1,25-dihydroxy-3-epi-20-epi-16-ene vitamin D.sub.3, and derivatives
thereof.
5. A method of treating a disorder characterized by an aberrant
activity of a vitamin D.sub.3-responsive cell, comprising
administering to a subject an effective amount of a 3-epi vitamin
D.sub.3 compound having the formula (I) of claim 1, such that the
aberrant activity of the vitamin D.sub.3-responsive cell is
reduced.
6. The method of claim 5, wherein the 3-epi vitamin D.sub.3
compound has at least one improved biological property compared to
vitamin D.sub.3 under the same conditions.
7. The method of claim 6, wherein the at least one improved
biological property comprises a reduction in hypercalcemia compared
to the hypercalcemia induced by vitamin D.sub.3 under the same
conditions.
8. The method of claim 6, wherein the at least one improved
biological property comprises an enhanced stability of the 3-epi
vitamin D.sub.3 compound compared to vitamin D.sub.3 under the same
conditions.
9. The method of claim 5, wherein the disorder comprises an
aberrant activity of a hyperproliferative skin cell.
10. The method of claim 9, wherein the disorder is selected from
the group consisiting of psoriasis, basal cell carcinoma and
keratosis.
11. The method of claim 5, wherein the disorder comprises an
aberrant activity of an endocrine cell.
12. The method of claim 11, wherein the endocrine cell is a
parathyroid cell and the aberrant activity is processing and/or
secretion of parathyroid hormone.
13. The method of claim 11, wherein the disorder is secondary
hyperparathyroidism.
14. The method of claim 5, wherein the disorder comprises an
aberrant activity of a bone cell.
15. The method of claim 14, wherein the disorder is selected from
the group consisiting of osteoporosis, osteodystrophy, senile
osteoporosis, osteomalacia, rickets, osteitis fibrosa cystica,
renal osteodystrophy, secondary hyperparathyrodism, cirrhosis, and
chronic renal disease.
16. The method of claim 5, wherein the subject is a mammal.
17. The method of claim 16, wherein the mammal is a human.
18. A method of reducing the activity of a hyperproliferative skin
cell, comprising administering to a subject a 3-epi vitamin D.sub.3
compound of claim 1, such that reduction of the hyperproliferative
skin cell activity occurs.
19. A method of ameliorating a deregulation in the activity of a
parathyroid cell, comprising administering to a subject a
therapeutically effective amount of a 3-epi vitamin D.sub.3
compound of claim 1 so as to ameliorate the deregulation of the
parathyroid cell activity.
20. A method of ameliorating a deregulation of calcium and
phosphate metabolism, comprising administering to a subject a
therapeutically effective amount of a 3-epi vitamin D.sub.3
compound of claim 1 so as to ameliorate the deregulation of the
calcium and phosphate metabolism.
21. The method of claim 20, wherein the deregulation of the calcium
and phosphate metabolism leads to osteoporosis.
22. A pharmaceutical composition comprising, a therapeutically
effective amount of a 3-epi vitamin D.sub.3 compound of claim 1 and
a pharmaceutically acceptable carrier.
23. The composition of claim 22, which is suitable for topical
administration.
24. The composition of claim 22, which is suitable for oral
administration.
25. A packaged compound, comprising a 3-epi vitamin D.sub.3
compound of claim 1 packaged with instructions for use of the
compound for treating a disorder characterized by an aberrant
activity of a vitamin D.sub.3-responsive cell.
26. A method of converting a 3-.beta. vitamin D.sub.3 compound into
its 3-epimer form by treating a cell having 3-.beta.-hydroxy
epimerase activity.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Application No. 60/046,643 filed on May 16, 1997, the
contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The importance of the vitamin D in the biological systems of
higher animals has been recognized since its discovery by Mellanby
in 1920 (Mellanby, E. (1921) Spec. Rep. Ser. Med. Res. Council (GB)
SRS 61:4). It was in the interval of 1920-1930 that vitamin D
officially became classified as a "vitamin" that was essential for
the normal development of the skeleton and maintenance of calcium
and phosphorous homeostasis.
[0003] Studies involving the metabolism of vitamin D.sub.3
(cholecalciferol) were initiated with the discovery and chemical
characterization of the plasma metabolite, 25-hydroxyvitamin
D.sub.3 [25(OH)D.sub.3] (Blunt, J. W. et al. (1968) Biochemistry
6:3317-3322) and the hormonally active form,
1.alpha.,25(OH).sub.2D.sub.3 (Myrtle, J. F. et al. (1970) J. Biol.
Chem. 245:1190-1196; Norman, A. W. et al. (1971) Science 173:51-54;
Lawson, D. E. M. et al (1971) Nature 230:228-230; Holick, M. F.
(1971) Proc. Natl. Acad. Sci. USA 68:803-804). The formulation of
the concept of a vitamin D endocrine system was dependent both upon
appreciation of the key role of the kidney in producing 1.alpha.,
25(OH).sub.2D.sub.3 in a carefully regulated fashion (Fraser, D. R.
and Kodicek, E (1970) Nature 288:764-766; Wong, R. G. et al. (1972)
J. Clin. Invest. 51:1287-1291), and the discovery of a nuclear
receptor for 1.alpha.,25(OH).sub.2D.sub.3 (VD.sub.3R) in the
intestine (Haussler, M. R. et al. (1969) Exp. Cell Res. 58:234-242;
Tsai, H. C. and Norman, A. W. (1972) J. Biol. Chem. 248:5967-5975).
The operation of the vitamin D endocrine system depends on the
following: first, on the presence of cytochrome P450 enzymes in the
liver (Bergman, T. and Postlind, H. (1991) Biochem. J. 276:427-432;
Ohyama, Y and Okuda, K. (1991) J. Biol. Chem. 266:8690-8695) and
kidney (Henry, H. L. and Norman, A. W. (1974) J. Biol. Chem.
249:7529-7535; Gray, R. W. and Ghazarian, J. G. (1989) Biochem. J.
259:561-568), and in a variety of other tissues to effect the
conversion of vitamin D.sub.3 into biologically active metabolites
such as 1.alpha.,25(OH).sub.2D.sub.3 and 24R,25(OH).sub.2D.sub.3;
second, on the existence of the plasma vitamin D binding protein
(DBP) to effect the selective transport and delivery of these
hydrophobic molecules to the various tissue components of the
vitamin D endocrine system (Van Baelen, H. et al. (1988) Ann NY
Acad. Sci. 538:60-68; Cooke, N. E. and Haddad, J. G. (1989) Endocr.
Rev. 10:294-307; Bikle, D. D. et al. (1986) J. Clin. Endocrinol.
Metab. 63:954-959); and third, upon the existence of
stereoselective receptors in a wide variety of target tissues that
interact with the agonist 1.alpha.,25(OH).sub.2D.sub.3 to generate
the requisite specific biological responses for this secosteroid
hormone (Pike, J. W. (1991) Annu. Rev. Nutr. 11:189-216). To date,
there is evidence that nuclear receptors for
1.alpha.,25(OH).sub.2D.sub.3 (VD.sub.3R) exist in more than 30
tissues and cancer cell lines (Reichel, H. and Norman, A. W. (1989)
Annu. Rev. Med. 40:71-78).
[0004] Vitamin D.sub.3 and its hormonally active forms are
well-known regulators of calcium and phosphorous homeostasis. These
compounds are known to stimulate, at least one of, intestinal
absorption of calcium and phosphate, mobilization of bone mineral,
and retention of calcium in the kidneys. Furthermore, the discovery
of the presence of specific vitamin D receptors in more than 30
tissues has led to the identification of vitamin D.sub.3 as a
pluripotent regulator outside its classical role in calcium/bone
homeostasis. A paracrine role for 1.alpha.,25(OH).sub.2D.sub- .3
has been suggested by the combined presence of enzymes capable of
oxidizing vitamin D.sub.3 into its active forms, e.g.,
25-OHD-1.alpha.-hydroxylase, and specific receptors in several
tissues such as bone, keratinocytes, placenta, and immune cells.
Moreover, vitamin D.sub.3 hormone and active metabolites have been
found to be capable of regulating cell proliferation and
differentiation of both normal and malignant cells (Reichel, H. et
al. (1989) Ann. Rev. Med. 40: 71-78).
[0005] Given the pluripotent activities of vitamin D.sub.3 and its
metabolites, much attention has focused on the development of
synthetic analogs of these compounds. A large number of these
analogs have involved structural modifications in the A ring, B
ring, C/D rings, and, primarily, the side chain (Bouillon, R. et
al., Endocrine Reviews 16(2):201-204). Although a vast majority of
the vitamin D.sub.3 analogs developed to date have involved
structural modifications in the side chain, a few studies have
reported the biological profile of A-ring diastereomers (Norman, A.
W. et al. J. Biol. Chem. 268 (27): 20022-20030). Despite much
effort in developing synthetic analogs, clinical applications of
vitamin D.sub.3 and its structural analogs have been limited by the
undesired side effects elicited by these compounds after
administration to a subject, such as the deregulation of calcium
and phosphorous homeostasis in vivo that results in
hypercalcemia.
SUMMARY OF THE INVENTION
[0006] The present invention is based, at least in part, on the
discovery of 3-epi vitamin D.sub.3 compounds having the orientation
of the hydroxy attached to the carbon at position 3 of the A-ring
of vitamin D.sub.3 inverted from a beta (.beta.) to an alpha
(.alpha.) configuration, and which are represented by the general
formula I described below. The 3-epi vitamin D.sub.3 compounds of
formula I are useful in treating disorders involving an aberrant
activity of hyperproliferative cells, e.g., hyperproliferative skin
cells, parathyroid cells and bone cells. These 3-epi forms of
vitamin D.sub.3 were first identified as metabolites of vitamin
D.sub.3 compounds produced via a tissue-specific pathway which
catalyzes the 3-.beta.-hydroxy epimerization of vitamin D.sub.3.
Isolated 3-epimer forms of vitamin D.sub.3 compounds have been
characterized and shown to have improved biological properties
compared to their isomeric counterparts, such as reduced
hypercalcemic activity and enhanced stability in vivo. The 3-epi
vitamin D.sub.3 compounds of the present invention can be used as
substitutes for natural and synthetic forms of vitamin D.sub.3, and
thus, these compounds provide for a safer alternative to
conventional therapeutic approaches.
[0007] Accordingly, the present invention pertains to isolated
3-epi vitamin D.sub.3 compounds represented by the general formula
(I): 1
[0008] wherein the orientation of the OH groups on the A-ring is in
an .alpha.-configuration; A and C can be a single or a double bond;
B can be a single, a double, e.g., E- or Z-double, or a triple
bond; R.sub.1 and R.sub.2 can, e.g., be chosen individually from
the group of: a hydrogen and a lower alkyl, e.g., a C.sub.1-C.sub.4
alkyl; R.sub.3 and R.sub.4 can, e.g., be chosen individually from
the group of: a lower alkyl, e.g., a C.sub.1-C.sub.4 alkyl, a
hydroxyalkyl, and a haloalkyl, e.g., a fluoroalkyl; X can be a
hydrogen or a hydroxy; and, Y can be a hydrogen, a hydroxy or an
oxygen atom (an oxo group), provided that the compound is not
1.alpha.,25(OH).sub.2-3-epi-D.sub.3.
[0009] In another aspect, the present invention further pertains to
a pharmaceutical composition containing, a therapeutically
effective amount of an isolated 3-epi vitamin D.sub.3 compound
having the above-described general formula (I) and a
pharmaceutically acceptable carrier.
[0010] The 3-epi vitamin D3 compounds of formula I can be
synthesized by perfusing a 3.beta.-vitamin D.sub.3 precursor, e.g.,
a vitamin D.sub.3 compound having the orientation of the hydroxy
group at position 3 of the A-ring in a .beta.-configuration, in a
tissue or a cell having 3.beta.-hydroxy epimerase activity, e.g., a
tissue or a cell containing an enzyme which catalyzes the
3-.beta.-hydroxy epimerization of these compounds Preferred cells
include keratinocytes, parathyroid cells, and bone cells.
Alternatively, the 3-epi vitamin D3 compounds of formula I can be
chemically synthesized.
[0011] In yet another aspect, this invention provides a method of
modulating a biological activity of a vitamin D.sub.3-responsive
cell. The method involves contacting the cell with an effective
amount of an isolated 3-epi vitamin D.sub.3 compound having the
above-described general formula (I) such that modulation of the
activity of the cell occurs.
[0012] Another aspect of the invention provides a method of
treating in a subject, a disorder characterized by aberrant growth
or activity of a vitamin D.sub.3 responsive cell. The method
involves administering to the subject an effective amount of a
pharmaceutical composition of a 3-epi vitamin D.sub.3 compound
having the above-described general formula (I) such that the growth
or activity of the cell is reduced.
[0013] In a preferred embodiment, the 3-epi vitamin D.sub.3
compound used in treating the subject has improved biological
properties compared to its isomeric counterparts, such as enhanced
stability and/or reduced toxicity. Preferably, the enhanced
stability of the 3-epi vitamin D.sub.3 compounds in vivo allows the
treatment of a particular disease or condition at a lower dosage,
thus reducing undesired side effects. In addition, the reduced
toxicity can result from a reduction in the induction of
hypercalcemia in vivo compared to the hypercalcemia induced by
vitamin D.sub.3 under the same conditions. In certain embodiments,
reduced hypercalcemia results from the modulation of at least one
of intestinal calcium transport, bone calcium metabolism and/or
gene expression, e.g., osteocalcin and/or calbindin synthesis.
[0014] In one aspect, a method for inhibiting the proliferation
and/or inducing the differentiation of a hyperproliferative skin
cell is provided, wherein the hyperproliferative skin cell is
selected from a group consisting of an epidermal cell and an
epithelial cell. Accordingly, therapeutic methods for treating
hyperproliferative skin disorders are provided.
[0015] In another aspect, the present invention demonstrates that
the isolated 3-epi vitamin D.sub.3 compounds of the present
invention supress secretion of a hormone in a vitamin D.sub.3
responsive cell, e.g., an endocrine cell responsive to vitamin
D.sub.3. In a preferred embodiment, the hormone is parathyroid
hormone (PTH). In certain embodiments, a method for inhibiting PTH
secretion in parathyroid cells using 3-epi vitamin D.sub.3
compounds is provided. Furthermore, therapeutic methods for
treating secondary hyperparathyroidism are also provided.
[0016] In yet another aspect, the 3-epi vitamin D.sub.3 compounds
of the present invention are useful in the treatment of disorder
characterized by a deregulation of calcium and phosphate
metabolism, comprising administering to a subject a pharmaceutical
preparation of a 3-epi vitamin D.sub.3 compound so as to ameliorate
the deregulation in calcium and phosphate metabolism.
[0017] In a preferred embodiment the disorder is osteoporosis. In
other embodiments, the 3-epi vitamin D.sub.3 compounds can be used
to treat diseases characterized by other deregulations in the
metabolism of calcium and phosphate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic representation of the pathways of
1.alpha.,25(OH).sub.2D.sub.3 metabolism through side chain
modification.
[0019] FIG. 2 is a schematic representation of the structure of
1.alpha.,25(OH).sub.2D.sub.3 and its A-ring diastereomers.
[0020] FIG. 3 is a schematic representation of the metabolism of
1.alpha.,25(OH).sub.2D.sub.3 via 3-epimerization.
[0021] FIG. 4 depicts the HPLC profile of standards of
1.alpha.,25(OH).sub.2D.sub.3 and their metabolites produced by
human keratinocytes.
[0022] FIG. 5 depicts the mass spectra of peak M produced in
keratinocytes (upper panel) and the synthetic of
1.alpha.,25(OH).sub.2D.sub.3 (lower panel).
[0023] FIG. 6 shows the straight phase and reverse phase HPLC
systems used to separate the four diastereomers of
1.alpha.,25(OH).sub.2D.sub.3.
[0024] FIG. 7 shows the HPLC profile and UV spectra of the
metabolites produced in human keratinocytes incubated with
1.alpha.,25(OH).sub.2-3-ep- i-D.sub.3. Elution position of
1.alpha.,25(OH).sub.2D.sub.3 is indicated by an asterisk (*).
[0025] FIG. 8A shows a detailed HPLC profile of the
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 metabolites produced in human
keratinocytes.
[0026] FIG. 8B summarizes the metabolism of
1.alpha.,25(OH).sub.2-3-epi-D.- sub.3 through A ring
modification.
[0027] FIG. 9 shows HPLC profiles of metabolites produced by human
keratinocytes incubated with tritiated 25(OH)D.sub.3. Upper panel
(A) depicts the time course of the production of various tritiated
metabolites (peak at retention time 11-12 min represents
25(OH)D.sub.3; the peak at retention time 38-39 min represents
1.alpha.,25(OH).sub.2D.su- b.3 and the peak at 35-36 min represents
1.alpha.,25(OH).sub.2-3-epi-D.sub- .3). Lower panel (B) depicts the
production rates of both 1.alpha.,25(OH).sub.2D.sub.3 and its
epimer.
[0028] FIG. 10 shows HPLC profiles of metabolites produced by
bovine parathryoid cells incubated with
1.alpha.,25(OH).sub.2D.sub.3.
[0029] FIG. 11 shows HPLC profiles of 1.alpha.,25(OH).sub.2D.sub.3
metabolites produced in human placental explants incubated with 1
uM 1.alpha.,25(OH).sub.2D.sub.3. Placental explants produced
metabolites of both C-24 and C-23 oxidation pathways. No evidence
of 1.alpha.,25(OH).sub.2D.sub.3 production is noted at the elution
position of the standard 1.alpha.,25(OH).sub.2-3-epi-D.sub.3.
[0030] FIG. 12 shows a comparison of the metabolism of
1.alpha.,25(OH).sub.2D.sub.3 in a cell line of immortalized human
keratinocytes (HACAT), a commonly studied cancer cell line (human
promyelocytic leukemic cell line, HL-60), and perfused rat kidney.
Primary cultures of human keratinocytes are shown as a control.
[0031] FIG. 13 shows the production of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in the rat osteosarcoma cell
line UMR 106.
[0032] FIG. 14 shows the HPLC profiles of
1.alpha.,25(OH).sub.2-3-epi-D.su- b.3 in rat osteosarcoma cells
(UMR-106) after 24 hours (upper panel) and 48 hours (lower panel)
of 1.alpha.,25(OH).sub.2D.sub.3 addition.
[0033] FIG. 15 shows the formation of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in a human osteosarcoma cell
(U-2 OS) grown at two different cell densities.
[0034] FIG. 16 shows the conversion of the vitamin D.sub.3 analog,
1.alpha.,25(OH).sub.2-16-ene-D.sub.3 into its 3-epi form in rat
osteosarcoma cell (UMR-106).
[0035] FIG. 17 shows the production of 3 epi forms of
1.alpha.,25(OH).sub.2-20-epi-D.sub.3 and
1.alpha.,25(OH).sub.2-16-ene-20-- epi-D.sub.3 in the rat
osteosarcoma cell (UMR-106).
[0036] FIG. 18 summarizes the HPLC profiles of vitamin D.sub.3
analogs tested in rat osteosarcoma cells (UMR-106). This summary
indicates that all of the vitamin D.sub.3 analogs tested are
converted into less polar 3-epi metabolites.
[0037] FIG. 19 shows the metabolism of
1.alpha.,25(OH).sub.2-16-ene-D.sub.- 3 and
1.alpha.,25(OH).sub.2-16-ene-23-yne-D.sub.3 into their 3 epi forms
in the rat osteosarcoma cell (UMR-106).
[0038] FIG. 20 depicts the biological activities of
1.alpha.,25(OH).sub.2D.sub.3 and
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in keratinocytes and bovine
parathyroid cells. Panel A shows the inhibitory effect of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 on keratinocyte cell growth
compared with 1.alpha.,25(OH).sub.2D.sub.3. Panel B shows the
inhibition of PTH secretion in bovine parathyroid cells by
1.alpha.,25(OH).sub.2-3-e- pi-D.sub.3 compared with
1.alpha.,25(OH).sub.2D.sub.3.
[0039] FIG. 21 is a schematic representation of the synthesis of
A-ring tritium labeled diastereomers of
1.alpha.,25(OH).sub.2D.sub.3.
[0040] FIG. 22 is a graph depicting a dose response of the
transcriptional activity of
1.alpha.,25(OH).sub.2-16-ene-23-yne-3-epi D.sub.3 and its isomeric
counterpart. The transcriptional activity of 1.alpha.,25(OH)2D3 is
shown for comparison.
[0041] FIG. 23A is a schematic representation of the synthesis of
[3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-Bis[[(1,1-dimethylethyl)-dimethylsilyl-
]oxy]-2-methylenecyclohexylidene]ethyl]diphenylphosphine oxide
(represented by the compound of formula II). The synthesis of the
compound of formula II was performed by a sequence of reactions
outlined in the Figure as Exp 1-11. The various starting materials
and intermediates used in the synthesis are identified as compounds
IV-XIV.
[0042] FIG. 23B is a representation of the chemical formulae of the
starting compounds used in the synthesis of 3-epi vitamin D3
compounds (represented by compounds of the formulae IIIb-i).
DETAILED DESCRIPTION OF THE INVENTION
[0043] The language "3-epi vitamin D.sub.3" or "3-epi D.sub.3"
compounds is intended to include vitamin D.sub.3 compounds having
the hydroxyl group, attached to the carbon at position 3 of the
A-ring in an .alpha.-configuration rather than a
.beta.-configuration, and which are represented by the general
formula I as described in detail below. These 3-epi forms of
vitamin D.sub.3 were first identified as metabolites of vitamin
D.sub.3 compounds produced in a tissue-specific manner.
[0044] As used herein, the language "tissue-specific" refers to a
novel pathway which catalyzes the 3-.beta.-hydroxy epimerization of
vitamin D.sub.3 in certain tissues,e.g., keratinocytes, parathyroid
cells, bone cells, but not in others, such as breast cancer cells
or leukemic cells. This novel pathway may be catalyzed by a single
enzyme or a combination of two or more enzymes referred to herein
as "3.beta.-hydroxy epimerase". The efficiency of the epimerization
reaction in a cell may vary depending on the differentiation state
of that cell. For example, epimerization of vitamin D.sub.3
compounds in vivo may occur more efficiently in differentiating
cells relative to actively proliferating cells.
[0045] 1.alpha.,25(OH).sub.2D.sub.3 is a hormonally active
secosteroid. The term "secosteroids" is art-recognized and includes
compounds in which one of the cyclopentanoperhydro-phenanthrene
rings of the steroid ring structure is broken. In the case of
vitamin D.sub.3, the 9-10 carbon-carbon bond of the B-ring is
broken, generating a seco-B-steroid. The official IUPAC name for
vitamin D.sub.3 is 9,10-secocholesta-5,7,10(1- 9)-trien-3B-ol. For
convenience, a 6-s-trans conformer of 1.alpha.,25(OH).sub.2D.sub.3
is illustrated herein having all carbon atoms numbered using
standard steroid notation. 2
[0046] In the formulas presented herein, the various substituents
are illustrated as joined to the steroid nucleus by one of these
notations: a dotted line (----) indicating a substituent which is
in the .beta.-orientation (i.e., above the plane of the ring), a
wedged solid line () indicating a substituent which is in the
.alpha.-orientation (i.e., below the plane of the molecule), or a
solid line (--) indicating a substituent in the plane of the ring.
It should be understood that the stereochemical convention in the
vitamin D field is opposite from the general chemical field,
wherein a dotted line indicates a substituent which is in an
.alpha.-orientation (i.e., below the plane of the molecule), and a
wedged solid line indicates a substituent which is in the
.beta.-orientation (i.e., above the plane of the ring). As shown,
the A ring of the hormone 1.alpha.,25(OH).sub.2D.sub.3 contains two
asymetric centers at chiral carbons-1 and -3, each one containing a
hydroxyl group in well-characterized configurations, namely the
1.alpha.- and 3.beta.-hydroxyl groups.
[0047] The vitamin D.sub.3 compounds of the present invention are
represented by the general formula (I): 3
[0048] wherein the orientation of the OH groups on the A-ring is in
an .alpha.-configuration; A and C can be a single or a double bond;
B can be a single or a double, e.g., E or Z-double, or a triple
bond; R.sub.1 and R.sub.2 can, e.g., be chosen individually from
the group of: a hydrogen and a lower alkyl, e.g., a C.sub.1-C.sub.4
alkyl; R.sub.3 and R.sub.4 can, e.g., be chosen individually from
the group of: a lower alkyl, e.g., a C.sub.1-C.sub.4 alkyl, a
hydroxyalkyl and a haloalkyl, e.g., a fluoroalkyl; X can be a
hydrogen or a hydroxy; and, Y can be a hydrogen, a hydroxy or an
oxygen atom (an oxo group), provided that the compound is not
1.alpha.,25(OH).sub.2-3-epi-D.sub.3.
[0049] In another preferred embodiment, A is a double bond, and B
is a triple bond.
[0050] In a preferred embodiment, the 3-epimer compounds of the
present invention are selected from the group consisting of
1.alpha.,25(OH).sub.2-3-epi-16-ene-D.sub.3,
1.alpha.,25(OH).sub.2-3-epi-1- 6-ene-23-yne-D3, 1,25
dihydroxy-24-oxo-3-epi-16-ene vitamin D.sub.3, and 1,24,25
trihydroxy-3-epi-16-ene-vitamin D.sub.3, each represented by the
formula: 4
[0051] respectively.
[0052] Additional preferred synthetic 3-epi analogs include
1,25-dihydroxy-3-epi-23-yne-vitamin D.sub.3,
1,25-dihydroxy-3-epi-20-epi-- vitamin D.sub.3, and
1,25-dihydroxy-3-epi-20-epi-16-ene vitamin D.sub.3, and derivatives
thereof. The chemical structures of these compounds prior to 3-epi
conversion are shown in FIGS. 17-20.
[0053] The term "epimer" or "epi" compounds is intended to include
compounds having a chiral carbon that varies in the orientation of
a single bond to a substituent on that carbon compared to the
naturally-occurring (or reference) compound, for example, a carbon
where the orientation of the bond to the substituent is in an
.alpha.-configuration, instead of a .beta.-configuration. The
3-epimer form of vitamin D.sub.3 having the general formula I has a
hydroxyl group attached to the carbon at position 3 of the A-ring
in an .alpha.-configuration rather than a .beta.-configuration,
whereas all other substituents can be in either an .alpha.- or a
.beta.-configuration.
[0054] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner. The term
"stereoisomers" or "isomers" refer to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space. In particular,
"enantiomers" refer to two stereoisomers of a compound which are
non-superimposable mirror images of one another. An equimolar
mixture of two enantiomers is called a "racemic mixture" or a
"racemate". "Diastereomers" refer to stereoisomers with two or more
centers of dissymmetry and whose molecules are not mirror images of
one another. With respect to the nomenclature of a chiral center,
terms "d" and "l" configuration are as defined by the IUPAC
Recommendations. As to the use of the terms, diastereomer,
racemate, epimer and enantiomer will be used in their normal
context to describe the stereochemistry of preparations.
[0055] As used herein, the language "isomeric counterparts of
vitamin D.sub.3" or "non-epimeric forms" refers to stereoisomers of
the 3-epi vitamin D.sub.3 compounds. For example, vitamin D.sub.3
compounds which have the orientation of the 3-hydroxy group in a
.beta.-configuration.
[0056] The terms "isolated" or "substantially purified" as used
interchangeably herein refer to vitamin D.sub.3 compounds in a
non-naturally occurring state. The compounds can be substantially
free of cellular material or culture medium when naturally
produced, or chemical precursors or other chemicals when chemically
synthesized. In other preferred embodiments, the terms "isolated"
or "substantially purified" also refer to preparations of a chiral
compound which substantially lack one of the enantiomers, i.e.,
enantiomerically enriched or non-racemic preparations of a
molecule. Similarly, isolated epimers or diasteromers refers to
preparations of chiral compounds which are substantially free of
other stereochemical forms. For instance, isolated or substantially
purified vitamin D.sub.3 compounds includes synthetic or natural
preparations of a vitamin D.sub.3 enriched for the stereoisomers
having a substituent attached to the chiral carbon at position 3 of
the A-ring in an .alpha.-configuration, and thus substantially
lacking other isomers having a .beta.-configuration. Unless
otherwise specified, such terms refer to vitamin D.sub.3
compositions in which the ratio of .alpha. to .beta. forms is
greater that 1:1 by weight. For instance, an isolated preparation
of an a epimer means a preparation having greater than 50% by
weight of the .alpha.-epimer relative to the .beta. stereoisomer,
more preferably at least 75% by weight, and even more preferably at
least 85% by weight. Of course the enrichment can be much greater
than 85%, providing a "substantially epimer enriched", which refers
to preparations of a compound which have greater than 90% of the
.alpha.-epimer relative to the .beta. stereoisomer, and even more
preferably greater than 95%. The term "substantially free of the
.beta. stereoisomer" will be understood to have similar purity
ranges.
[0057] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six,
and most preferably from one to four carbon atoms in its backbone
structure, which may be straight or branched-chain. Examples of
lower alkyl groups include methyl, ethyl, n-propyl, i-propyl,
tert-butyl, hexyl, heptyl, octyl and so forth. In preferred
embodiment, the term "lower alkyl" includes a straight chain alkyl
having 4 or fewer carbon atoms in its backbone, e.g.,
C.sub.1-C.sub.4 alkyl.
[0058] Moreover, the term alkyl as herein is intended to include
both "unsubstituted alkyls" and "substituted alkyls", the latter of
which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such
substituents can include, for example, halogen (including
fluoroalkyl), hydroxyl (including hydroxyalkyl).
[0059] As used herein, the term "halogen" designates --F, --Cl,
--Br or --I; the term "sulfhydryl" or "thiol" means --SH; the term
"hydroxyl" means --OH.
[0060] Vitamin D.sub.3 Synthesis
[0061] The 3-epi vitamin D.sub.3 compounds of the present invention
can be prepared by enzymatic conversion of a 3.beta.-vitamin
D.sub.3 precursor, e.g., by perfusing a 3.beta.-vitamin D.sub.3
precursor, e.g., a vitamin D3 compound having the orientation of
the hydroxy group at position 3 of the A-ring in a
.beta.-configuration, in a tissue-containing an enzyme which
catalyzes the epimerization of the 3-.beta.-hydroxyl group to the
3.alpha. form vitamin D.sub.3 compounds, e.g., keratinocytes,
parathyroid cells, bone cells, as described in Examples I-IV,
VII-IX and XI-XIII.
[0062] Alternatively, the 3-epi vitamin D.sub.3 compounds of
formula I can be synthesized chemically using a variety of
synthetic methods. For example, 3-epi vitamin D.sub.3 compounds of
formula I can be formed by a convergent synthesis summarized in
FIG. 23A. Briefly, as detailed in Examples XVIII-XXV and
illustrated in FIG. 23A, 3-epi compounds of the invention can be
prepared by reacting an anion corresponding to
[3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-Bis[[(1,1-dimethylethyl)-dimethylsilyl-
]oxy]-2-methylenecyclohexylidene]ethyl]diphenylphosphine oxide
(referred to herein as the compound of formula II) with
n-butyllithium at -78.degree. C. in anhydrous tetrahydrofuran, with
a starting compound (for example, a compound represented by the
formulae IIIb-i of FIG. 23B), followed by removal of the protecting
silyl groups with tetra-n-butylammonium fluoride in tetrahydrofuran
at room temperature. The synthesis of the compound of formula II
can be performed by a sequence of reactions as summarized in Exp.
1-11 outlined in FIG. 23A and detailed in Example XVIII.
[0063] The above-described synthetic scheme can be summarized in
the following reaction: 5
[0064] wherein A can be a single or a double bond; B can be single,
double, e.g., E or Z-double, or a triple bond; and R.sub.1 and
R.sub.2 can be a hydrogen or a lower alkyl, e.g., a C.sub.1-C.sub.4
alkyl; R.sub.3 and R.sub.4 can be a lower alkyl, e.g., a
C.sub.1-C.sub.4 alkyl, a hydroxyalkyl or a haloalkyl, e.g., a
fluoroalkyl.
[0065] Naturally occurring or synthetic isomers can be separated in
several ways known in the art. Examples of straight phase and
reverse phase HPLC systems used to separate natural or synthetic
diastereomers of 1.alpha.,25(OH).sub.2D.sub.3 are detailed in the
appended examples and illustrated in FIG. 7. Further methods for
separating a racemic mixture of two enantiomers include
chromatography using a chiral stationary phase (see, e.g., "Chiral
Liquid Chromatography", W. J. Lough, Ed. Chapman and Hall, New York
(1989)). Enantiomers can also be separated by classical resolution
techniques. For example, formation of diastereomeric salts and
fractional crystallization can be used to separate enantiomers. For
the separation of enantiomers of carboxylic acids, the
diastereomeric salts can be formed by addition of enantiomerically
pure chiral bases such as brucine, quinine, ephedrine, strychnine,
and the like. Alternatively, diastereomeric esters can be formed
with enantiomerically pure chiral alcohols such as menthol,
followed by separation of the diastereomeric esters and hydrolysis
to yield the free, enantiomerically enriched carboxylic acid. For
separation of the optical isomers of amino compounds, addition of
chiral carboxylic or sulfonic acids, such as camphorsulfonic acid,
tartaric acid, mandelic acid, or lactic acid can result in
formation of the diastereomeric salts.
[0066] Pharmaceutical Compositions
[0067] In another aspect, the present invention provides
pharmaceutically acceptable compositions which comprise a
therapeutically-effective amount of one or more of the isolated
3-epi vitamin D.sub.3 compounds of formula I, formulated together
with one or more pharmaceutically acceptable carrier(s).
[0068] In a preferred embodiment, these pharmaceutical compositions
are suitable for topical or oral administration to a subject. In
other embodiments, as described in detail below, the pharmaceutical
compositions of the present invention may be specially formulated
for administration in solid or liquid form, including those adapted
for the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets,
boluses, powders, granules, pastes; (2) parenteral administration,
for example, by subcutaneous, intramuscular or intravenous
injection as, for example, a sterile solution or suspension; (3)
topical application, for example, as a cream, ointment or spray
applied to the skin; (4) intravaginally or intrarectally, for
example, as a pessary, cream or foam; or (5) aerosol, for example,
as an aqueous aerosol, liposomal preparation or solid particles
containing the compound.
[0069] In certain embodiments, the subject is a mammal, e.g., a
primate, e.g., a human. As used herein, the language "subject" is
intended to include human and non-human animals. Preferred human
animals include a human patient having a disorder characterized by
the aberrant activity of a vitamin D.sub.3-responsive cell. The
term "non-human animals" of the invention includes all vertebrates,
e.g., mammals and non-mammals, such as non-human primates, sheep,
dog, cow, chickens, amphibians, reptiles, etc.
[0070] The phrase "therapeutically-effective amount" as used herein
means that amount of a 3-epi vitamin D.sub.3 compound(s) of formula
I, or composition comprising such a compound which is effective for
the 3-epi compound to produce its intended function, e.g., the
modulation of activity of a vitamin D.sub.3-response cell. The
effective amount can vary depending on such factors as the type of
cell growth being treated or inhibited, the particular type of
3-epi vitamin D.sub.3 compound, the size of the subject, or the
severity of the undesirable cell growth or activity. One of
ordinary skill in the art would be able to study the aforementioned
factors and make the determination regarding the effective amount
of the 3-epi vitamin D.sub.3 compound of formula I without undue
experimentation.
[0071] In certain embodiments, one or more 3-epi vitamin D.sub.3
compounds as represented by formula I may be administered alone, or
as part of combinatorial therapy. For example, the 3-epi vitamin
D.sub.3 compounds can be conjointly administered with one or more
agents such as mitotic inhibitors, alkylating agents,
antimetabolites, nucleic acid, intercalating agents, topoisomerase
inhibitors, agents which promote apoptosis, and/or agents which
modulate immune responses. The effective amount of 3-epi vitamin
D.sub.3 compound used can be modified according to the
concentrations of the other agents used.
[0072] In vitro assay as described in Example XIV below using
keratinocytes or parathyroid cells, or an assay similar thereto
(e.g., differing in choice of cells, e.g., bone cells, intestinal
cells, neoplastic cells) can be used to determine an "effective
amount" of the 3-epi vitamin D.sub.3 compounds, or combinations
thereof The ordinarily skilled artisan would select an appropriate
amount of each individual compound in the combination for use in
the aforementioned in vitro assay or similar assays. Changes in
cell activity or cell proliferation can be used to determine
whether the selected amounts are "effective amount" for the
particular combination of compounds. The regimen of administration
also can affect what constitutes an effective amount. As described
in detail below, 3-epi vitamin D.sub.3 compounds of formula I can
be administered to the subject prior to, simultaneously with, or
after the administration of the other agent(s). Further, several
divided dosages, as well as staggered dosages, can be administered
daily or sequentially, or the dose can be proportionally increased
or decreased as indicated by the exigencies of the therapeutic
situation.
[0073] The phrase "pharmaceutically acceptable" is employed herein
to refer to those 3-epi vitamin D.sub.3 compounds of formula I,
compositions containing such compounds, and/or dosage forms which
are, within the scope of sound medical judgment, suitable for use
in contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk
ratio.
[0074] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject chemical from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0075] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0076] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0077] Compositions containing the 3-epi vitamin D.sub.3 compounds
of the present invention include those suitable for oral, nasal,
topical (including buccal and sublingual), rectal, vaginal, aerosol
and/or parenteral administration. The compositions may conveniently
be presented in unit dosage form and may be prepared by any methods
well known in the art of pharmacy. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will vary depending upon the host being treated, the
particular mode of administration. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will generally be that amount of the compound which
produces a therapeutic effect. Generally, out of one hundred
percent, this amount will range from about 1 percent to about
ninety-nine percent of active ingredient, preferably from about 5
percent to about 70 percent, most preferably from about 10 percent
to about 30 percent.
[0078] Methods of preparing these compositions include the step of
bringing into association a 3-epi vitamin D.sub.3 compound(s) of
formula I with the carrier and, optionally, one or more accessory
ingredients. In general, the formulations are prepared by uniformly
and intimately bringing into association a 3-epi vitamin D.sub.3
compound with liquid carriers, or finely divided solid carriers, or
both, and then, if necessary, shaping the product.
[0079] Compositions of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a 3-epi vitamin
D.sub.3 compound(s) of formula I as an active ingredient. A
compound may also be administered as a bolus, electuary or
paste.
[0080] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically-acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, acetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay;
(9) lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof, and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0081] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered peptide or peptidomimetic moistened with an
inert liquid diluent.
[0082] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0083] Liquid dosage forms for oral administration of the 3-epi
vitamin D.sub.3 compound(s) of the invention include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
ingredient, the liquid dosage forms may contain inert diluents
commonly used in the art, such as, for example, water or other
solvents, solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof.
[0084] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0085] Suspensions, in addition to the active 3-epi vitamin D.sub.3
compound(s) of formula I may contain suspending agents as, for
example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol
and sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite, agar-agar and tragacanth, and mixtures
thereof.
[0086] Pharmaceutical compositions of the invention for rectal or
vaginal administration may be presented as a suppository, which may
be prepared by mixing one or more 3-epi vitamin D.sub.3 compound(s)
of formula I with one or more suitable nonirritating excipients or
carriers comprising, for example, cocoa butter, polyethylene
glycol, a suppository wax or a salicylate, and which is solid at
room temperature, but liquid at body temperature and, therefore,
will melt in the rectum or vaginal cavity and release the active
agent.
[0087] Compositions of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0088] Dosage forms for the topical or transdermal administration
of a 3-epi vitamin D.sub.3 compound(s) of formula I include
powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches and inhalants. The active 3-epi vitamin D.sub.3
compound(s) of formula I may be mixed under sterile conditions with
a pharmaceutically-acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0089] The ointments, pastes, creams and gels may contain, in
addition to 3-epi vitamin D.sub.3 compound(s) of formula I,
excipients, such as animal and vegetable fats, oils, waxes,
paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, silicic acid, talc and zinc oxide,
or mixtures thereof.
[0090] Powders and sprays can contain, in addition to a 3-epi
vitamin D.sub.3 compound(s) of formula I, excipients such as
lactose, talc, silicic acid, aluminum hydroxide, calcium silicates
and polyamide powder, or mixtures of these substances. Sprays can
additionally contain customary propellants, such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,
such as butane and propane.
[0091] The 3-epi vitamin D.sub.3 compound(s) of formula I can be
alternatively administered by aerosol. This is accomplished by
preparing an aqueous aerosol, liposomal preparation or solid
particles containing the compound. A nonaqueous (e.g., fluorocarbon
propellant) suspension could be used. Sonic nebulizers are
preferred because they minimize exposing the agent to shear, which
can result in degradation of the compound.
[0092] Ordinarily, an aqueous aerosol is made by formulating an
aqueous solution or suspension of the agent together with
conventional pharmaceutically acceptable carriers and stabilizers.
The carriers and stabilizers vary with the requirements of the
particular compound, but typically include nonionic surfactants
(Tweens, Pluronics, or polyethylene glycol), innocuous proteins
like serum albumin, sorbitan esters, oleic acid, lecithin, amino
acids such as glycine, buffers, salts, sugars or sugar alcohols.
Aerosols generally are prepared from isotonic solutions.
[0093] Transdermal patches have the added advantage of providing
controlled delivery of a 3-epi vitamin D.sub.3 compound(s) of
formula I to the body. Such dosage forms can be made by dissolving
or dispersing the agent in the proper medium. Absorption enhancers
can also be used to increase the flux of the peptidomimetic across
the skin. The rate of such flux can be controlled by either
providing a rate controlling membrane or dispersing the
peptidomimetic in a polymer matrix or gel.
[0094] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0095] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more 3-epi vitamin
D.sub.3 compound(s) of formula I in combination with one or more
pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents.
[0096] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0097] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0098] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0099] Injectable depot forms are made by forming microencapsule
matrices of 3-epi vitamin D.sub.3 compound(s) of formula I in
biodegradable polymers such as polylactide-polyglycolide. Depending
on the ratio of drug to polymer, and the nature of the particular
polymer employed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include poly(orthoesters)
and poly(anhydrides). Depot injectable formulations are also
prepared by entrapping the drug in liposomes or microemulsions
which are compatible with body tissue.
[0100] When the 3-epi vitamin D.sub.3 compound(s) of the present
invention are administered as pharmaceuticals, to humans and
animals, they can be given per se or as a pharmaceutical
composition containing, for example, 0.1 to 99.5% (more preferably,
0.5 to 90%) of active ingredient in combination with a
pharmaceutically acceptable carrier.
[0101] The term "administration," is intended to include routes of
introducing a subject the 3-epimer vitamin D.sub.3 compound of
formula I to perform their intended function. Examples of routes of
administration which can be used include injection (subcutaneous,
intravenous, parenterally, intraperitoneally, intrathecal, etc.),
oral, inhalation, rectal and transdermal. The pharmaceutical
preparations are of course given by forms suitable for each
administration route. For example, these preparations are
administered in tablets or capsule form, by injection, inhalation,
eye lotion, ointment, suppository, etc. administration by
injection, infusion or inhalation; topical by lotion or ointment;
and rectal by suppositories. Oral administration is preferred. The
injection can be bolus or can be continuous infusion. Depending on
the route of administration, the 3-epi vitamin D.sub.3 compound of
formula I can be coated with or disposed in a selected material to
protect it from natural conditions which may detrimentally effect
its ability to perform its intended function. The 3-epi vitamin
D.sub.3 compound of formula I can be administered alone, or in
conjunction with either another agent as described above or with a
pharmaceutically acceptable carrier, or both. The 3-epi vitamin
D.sub.3 compound can be administered prior to the administration of
the other agent, simultaneously with the agent, or after the
administration of the agent. Furthermore, the 3-epi vitamin D.sub.3
compound can also be administered in a proform which is converted
into its active metabolite, or more active metabolite in vivo.
[0102] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticulare, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0103] The phrases "systemic administration," "administered
systemically", "peripheral administration" and "administered
peripherally" as used herein mean the administration of a 3-epi
vitamin D.sub.3 compound(s) of formula I, drug or other material,
such that it enters the patient's system and, thus, is subject to
metabolism and other like processes, for example, subcutaneous
administration.
[0104] These 3-epi vitamin D.sub.3 compound(s) may be administered
to a "subject", e.g., mammals, e.g., humans and other animals.
Administration can be carried out by any suitable route of
administration, including orally, nasally, as by, for example, a
spray, rectally, intravaginally, parenterally, intracisternally and
topically, as by powders, ointments or drops, including buccally
and sublingually.
[0105] Regardless of the route of administration selected, the
3-epi vitamin D.sub.3 compound(s) of formula I, which may be used
in a suitable hydrated form, and/or the pharmaceutical compositions
of the present invention, are formulated into
pharmaceutically-acceptable dosage forms by conventional methods
known to those of skill in the art.
[0106] Actual dosage levels and time course of administration of
the active ingredients in the pharmaceutical compositions of this
invention may be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic
response for a particular patient, composition, and mode of
administration, without being toxic to the patient. Exemplary dose
range is from 0.1 to 10 mg per day.
[0107] Uses of the Vitamin D.sub.3 Compounds of the Invention
[0108] Another aspect of the invention pertains to isolated 3-epi
vitamin D.sub.3 compounds of formula I having at least one
biological activity of vitamin D.sub.3, and having improved
biological properties when administered into a subject than vitamin
D.sub.3 under the same conditions, as well as, methods of testing
and using these compounds to treat disorders involving an aberrant
activity of hyperproliferative skin cells, parathyroid cells and
bone cells.
[0109] The language "biological activities" of vitamin D.sub.3 is
intended to include all activities elicited by vitamin D.sub.3
compounds in a responsive cell. This term includes genomic and
non-genomic activities elicited by these compounds (Bouillon, R. et
al. (1995) Endocrinology Reviews 16(2):206-207; Norman A. W. et al.
(1992) J. Steroid Biochem Mol. Biol 41:231-240; Baran D. T. et al.
(1991) J. Bone Miner Res. 6:1269-1275; Caffrey J. M. and
Farach-Carson M. C. (1989) J. Biol. Chem. 264:20265-20274; Nemere
I. et al. (1984) Endocrinology 115:1476-1483).
[0110] As used herein, the term "vitamin D.sub.3-responsive cell"
includes any cell which is capable of responding to a 3-epi vitamin
D.sub.3 compound of formula I and is associated with disorders
involving an aberrant activity of hyperproliferative skin cells,
parathyroid cells and bone cells. These cells can respond to
vitamin D.sub.3 activation by triggering genomic and/or non-genomic
responses that ultimately result in the modulation of cell
proliferation, differentiation survival, and/or other cellular
activities such as hormone secretion. In a preferred embodiment,
the ultimate responses of a cell are inhibition of cell
proliferation and/or induction of differentiation-specific genes.
Exemplary vitamin D.sub.3 responsive cells include bone cells,
endocrine cells, epidermal cells, endodermal cells, among
others.
[0111] As used herein, the language "vitamin D.sub.3 agonist"
refers to a compound which potentiates, induces or otherwise
enhances a biological activity of vitamin D.sub.3 in a responsive
cell. In certain embodiments, an agonist may induce a genomic
activity, e.g., activation of transcription by a vitamin D.sub.3
nuclear receptor, or a non-genomic vitamin D.sub.3 activity, e.g.,
potentiation of calcium channel activity. In other embodiments, the
agonist potentiates the sensitivity of the receptor to another
vitamin D.sub.3 compound, e.g., treatment with the agonist lowers
the concentration of vitamin D.sub.3 compound required to induce a
particular biological response. The language "vitamin D.sub.3
antagonist" is intended to include those compounds that oppose any
biological activity of a vitamin D.sub.3 compound.
[0112] The language "non-genomic" vitamin D.sub.3 activities
include cellular (e.g., calcium transport across a tissue) and
subcellular activities (e.g., membrane calcium transport opening of
voltage-gated calcium channels, changes in intracellular second
messengers) elicited by vitamin D.sub.3 compounds in a responsive
cell. Electrophysiological and biochemical techniques for detecting
these activities are known in the art. An example of a particular
well-studied non-genomic activity is the rapid hormonal stimulation
of intestinal calcium mobilization, termed "transcaltachia" (Nemere
I. et al. (1984) Endocrinology 115:1476-1483; Lieberherr M. et al.
(1989) J. Biol. Chem. 264:20403-20406; Wali R. K. et al. (1992)
Endocrinology 131:1125-1133; Wali R. K. et al. (1992) Am. J.
Physiol. 262:G945-G953; Wali R. K. et al. (1990) J. Clin. Invest.
85:1296-1303; Bolt M. J. G. et al. (1993) Biochem. J. 292:271-276).
Detailed descriptions of experimental transcaltachia are provided
in Norman, A. W. (1993) Endocrinology 268(27):20022-20030;
Yoshimoto, Y. and Norman, A. W. (1986) Endocrinology118:2300-2304.
Changes in calcium activity and second messenger systems are well
known in the art and are extensively reviewed in Bouillion, R. et
al. (1995) Endocrinology Review 16(2): 200-257; the description of
which is incorporated herein by reference.
[0113] Exemplary systems and assays for testing non-genomic
activity are extensively described in the following references,
liver (Baran D. T. et al. (1989) FEBS Lett 259:205-208; Baran D. T.
et al. (1990) J. Bone Miner Res. 5:517-524; rat osteoblasts, e.g.,
ROS 17/2.8 cells (Baran D. T. et al. (1991) J. Bone Miner Res.
6:1269-1275; Caffrey J. M. (1989) J. Biol. Chem. 264:20265-20274;
Civitelli R. et al. (1990) Endocrinology 127:2253-2262), muscle
(DeBoland A. R. and Boland R. L. (1993) Biochem. Biophys Acta Mol.
Cell Res. 1179:93-104; Morelli S. et al. (1993) Biochem J.
289:675-679; Selles J. and Boland R. L. (1991) Mol. Cell
Endocrinol. 82:229-235), and in parathyroid cells (Bourdeau A. et
al. (1990) Endocrinology 127:2738-2743).
[0114] The language "genomic" activities or effects of vitamin
D.sub.3 is intended to include those activities mediated by the
nuclear receptor for 1.alpha.,25(OH).sub.2D.sub.3 (VD.sub.3R),
e.g., transcriptional activation of target genes. The term
"VD.sub.3Rs" is intended to include members of the type II class of
steroid/thyroid superfamily of receptors (Stunnenberg, H. G. (1993)
Bio Essays 15(5):309-15), which are able to bind transactivate
through the vitamin D response element (VDRE) in the absence of a
ligand (Damm et al. (1989) Nature 339:593-97; Sap et al. Nature
343:177-180). As used herein "VDREs" refer to a DNA sequences
composed of half-sites arranged as direct repeats. It is known in
the art that type II receptors do not bind to their respective
binding site as homodimers but require an auxiliary factor, RXR
(e.g. RXR.alpha., RXR.beta., RXR.gamma.) for high affinity binding
Yu et al. (1991) Cell 67:1251-1266; Bugge et al. (1992) EMBO J.
11:1409-1418; Kliewer et al. (1992) Nature 355:446-449; Leid et al.
(1992) EMBO J. 11:1419-1435; Zhang et al. (1992) Nature
355:441-446).
[0115] Following binding, the transcriptional activity of a target
gene (i.e., a gene associated with the specific DNA sequence) is
enhanced as a function of the ligand bound to the receptor
heterodimer. Exemplary vitamin D.sub.3-responsive genes include
osteocalcin, osteopontin, calbindins, parathyroid hormone (PTH),
24-hydroxylase, and .alpha. .sub.v.beta.3-integrin. Genomic
activities elicited by 3-epi vitamin D.sub.3 compounds can be
tested by detecting the transcriptional upregulation of a vitamin
D.sub.3 responsive gene in a cell containing VD3R.sub.s as
illustrated in Example XVII below. For example, the steady state
levels of responsive gene mRNA or protein, e.g. calbindin gene,
osteocalcin gene, can be detected in vivo or in vitro. Suitable
cells that can be used include any vitamin D.sub.3 responsive cell,
e.g., keratinocytes, parathyroid cells, MG-63 cell line,
ROS-17/2.8, among others.
[0116] In accordance with a still further embodiment of the present
invention, convenient screening methods can be established in cell
lines containing VD.sub.3R.sub.s, comprising (i) establishing a
culture of these cells which include a reporter gene construct
having a reporter gene which is expressed in an VD.sub.3R-dependent
fashion; (ii) contacting these cells with 3-epi vitamin D.sub.3
compounds; and (iii) monitoring the amount of expression of the
reporter gene. Expression of the reporter gene reflects
transcriptional activity of the VD.sub.3R.sub.s protein. Typically,
the reporter gene construct will include a reporter gene in
operative linkage with one or more transcriptional regulatory
elements responsive to VD.sub.3R.sub.s, e.g., the VD.sub.3R.sub.s
response element (VDRE) known in the art. The amount of
transcription from the reporter gene may be measured using any
method known to those of skill in the art to be suitable. For
example, specific mRNA expression may be detected using Northern
blots or specific protein product may be identified by a
characteristic stain, immunoassay or an intrinsic activity. In
preferred embodiments, the gene product of the reporter is detected
by an intrinsic activity associated with that product. For
instance, the reporter gene may encode a gene product that, by
enzymatic activity, gives rise to a detection signal based on
color, fluorescence, or luminescence. The amount of expression from
the reporter gene is then compared to the amount of expression in
either the same cell in the absence of the test compound or it may
be compared with the amount of transcription in a substantially
identical cell that lacks the specific receptors. Agonistic vitamin
D.sub.3 compounds can then be readily detected by the increased
activity or concentration of these reporter genes relative to
untransfected controls.
[0117] After identifying certain test compounds as potential
agonists or antagonists of vitamin D.sub.3 compounds, the
practioner of the subject assay will continue to test the efficacy
and specificity of the selected compounds both in vitro and in
vivo. Whether for subsequent in vivo testing, or for administration
to an animal as an approved drug, agents identified in the subject
assay can be formulated in pharmaceutical preparations, such as
described above, for in vivo administration to an animal,
preferably a human.
[0118] As described herein, the 3-epi-vitamin D.sub.3 compounds of
the present invention show improved biological properties than
their isomeric counterparts. As used herein, the language "improved
biological properties" refers to any activity inherent in a 3-epi
vitamin D.sub.3 compound of formula I that enhances its
effectiveness in vivo. In a preferred embodiment, this term refers
to any qualitative or quantitative improved therapeutic property of
a vitamin D.sub.3 compound, such as enhanced stability in vivo
and/or reduced toxicity, e.g., reduced hypercalcemic activity. The
improved biological property may occur in both a tissue-specific
and non-specific manner. For example, certain tissues may be
capable of metabolizing 3-epi forms of vitamin D.sub.3 into unique
metabolites that enhance in a tissue-specific manner the biological
activities of this compound.
[0119] The increased stability of 3-epi vitamin D3 compounds is
demonstated below in tissue incubation studies which indicate that
in prolonged incubations, the concentration of
1.alpha.,25(OH).sub.2-3-epi-D- .sub.3 is significantly higher when
compared to the unmetabolized 1.alpha.,25(OH).sub.2D.sub.3
substrate (Examples IX, XV and XVI, as well as FIGS. 13 and 14).
These data indicate that 3-epi forms of vitamin D.sub.3 are more
stable in vivo compared to their isomeric counterparts. Any 3-epi
vitamin D.sub.3 compound that shows significantly higher
concentrations after prolonged incubations in vivo or in vitro, or
that shows an increase in the binding to plasma vitamin D binding
protein (DBP) compared to its isomeric counterpart is classified as
a compound having enhanced stability (See Examples IX, XV, XVI,
FIGS. 13 and 14, Table 1; see also, A. W. Norman et al. J. Biol.
Chem. 268 (27): 20022-20030).
[0120] Hypercalcemic conditions or deregulation of calcium
homeostasis have limited clinical applications of vitamin D.sub.3
analogs in the past. The present invention provides 3-epimeric
forms of vitamin D.sub.3 that, while retaining vitamin D.sub.3
biological activities, have reduced hypercalcemic activity. As
summarized in Example XV and Table 1 below, 3-epi vitamin D.sub.3
compounds exhibit reduced calcium mobilization activity in vivo as
exemplified by a marked decrease in intestinal calcium transport
(ICA) and bone calcium mobilization (BCM) when compared to their
non-epimeric counterparts. Thus, the dissociation of the biological
activities (cell differentiation, immune effects) from the reduced
deregulatory effect on calcium homeostasis provides 3-epi vitamin
D.sub.3 compounds having significant therapeutic advantages over
3.beta.-isomers of vitamin D.sub.3.
[0121] The language "reduced toxicity" is intended to include a
reduction in any undesired side effect elicited by a vitamin
D.sub.3 compound when administered in vivo, e.g., a reduction in
the hypercalcemic activity. The language "hypercalcemia" or
"hypercalcemic activity" is intended to have its accepted clinical
meaning, namely, increases in calcium serum levels that are
manifested in a subject by the following side effects, depression
of central and peripheral nervous system, muscular weakness,
constipation, abdominal pain, lack of appetite and, depressed
relaxation of the heart during diastole. Symptomatic manifestations
of hypercalcemia are triggered by a stimulation of at least one of
the following activities, intestinal calcium transport, bone
calcium metabolism and osteocalcin synthesis (reviewed in Boullion,
R. et al. (1995) Endocrinology Reviews 16(2): 200-257).
[0122] Compounds exhibiting reduced hypercalcemic activity can be
tested in vivo or in vitro using methods known in the art and
reviewed by Boullion, R. et al. (1995) Endocrinology Reviews 16(2):
200-257. For example, the serum calcium levels following
administration of a vitamin D.sub.3 compound can be tested by
routine experimentation (Lemire, J. M. (1994) Endocrinology
135(6):2818-2821). Briefly, 3-epi vitamin D.sub.3 compounds can be
administered intramuscularly to vitamin D.sub.3-deficient subjects,
e.g., rodents, e.g. mouse, or avian species, e.g. chick. At
appropriate time intervals, serum calcium levels and extent of
calcium uptake can be used to determine the level of bone calcium
mobilization (BCM) and intestinal calcium absorption (ICA) induced
by the tested vitamin D.sub.3 compound as illustrated in Table 1
below and described in Norman, A. W. et al. (1993) J. Biol. Chem.
268(27):20022-20029. Compounds which upon addition fail to increase
the concentration of calcium in the blood serum, thus showing
decreased BCM and ICA responses compared to their isomeric
counterparts, are considered to have reduced hypercalcemic
activity. Compounds which have reduced toxicity compared to their
isomeric counterparts are considered to have reduced toxicity.
Additional calcium homeostasis-related assays are described below
in the Calcium and Phosphate Homeostasis section.
[0123] Hyperproliferative Conditions
[0124] In another aspect the present invention provides a method of
treating in a subject, a disorder characterized by aberrant
activity of a vitamin D.sub.3-responsive cell. The method involves
administering to the subject an effective amount of a
pharmaceutical composition of a 3-epi vitamin D.sub.3 compound of
formula I such that the activity of the cell is modulated. As used
herein, the language "modulate" refers to increases or decreases in
the activity of a cell in response to exposure to a compound of the
invention, e.g., the inhibition of proliferation and/or induction
of differentiation of at least a sub-population of cells in an
animal such that a desired end result is achieved, e.g. a
therapeutic result. In preferred embodiments, this phrase is
intended to include hyperactive conditions that result in
pathological disorders.
[0125] In accordance with the present invention, 3-epi vitamin
D.sub.3 compounds of formula I can be used in the treatment of both
pathologic and non-pathologic proliferative conditions
characterized by unwanted growth of hyperproliferative skin cells.
In other embodiments, the cells to be treated are aberrant
secretory cells, e.g., parathyroid cells.
[0126] The use of vitamin D.sub.3 compounds in treating
hyperproliferative conditions has been limited because of their
hypercalcemic effects. As shown in Example XIV, the present
invention provides highly potent inhibitors of keratinocyte
proliferation, which show reduced hypercalcemic activity compared
to their isomeric counterparts. Thus, the 3-epi forms of vitamin
D.sub.3 compounds provides a less toxic alternative to current
methods of treatment.
[0127] In one embodiment, this invention features a method for
inhibiting the proliferation and/or inducing the differentiation of
a hyperproliferative skin cell, e.g., an epidermal or an epithelial
cell, e.g. a keratinocytes, by contacting the cells with a 3-epi
vitamin D.sub.3 compounds of formula I. In general, the method
includes a step of contacting a pathological or non-pathological
hyperproliferative cell with an effective amount of such 3-epi
vitamin D.sub.3 compound to promote the differentiation of the
hyperproliferative cells The present method can be performed on
cells in culture, e.g. in vitro or ex vivo, as shown in Example
XIV, or can be performed on cells present in an animal subject,
e.g., as part of an in vivo therapeutic protocol. The therapeutic
regimen can be carried out on a human or any other animal
subject.
[0128] The 3-epi-vitamin D.sub.3 compounds of the present invention
can be used to treat a hyperproliferative skin disorder. Examples
of these disorders include psoriasis, such as eczema; lupus
associated skin lesions; psoriatic arthritis; rheumatoid arthritis
that involves hyperproliferation and inflammation of
epithelial-related cells lining the joint capsule; basal cell
carcinoma; keratinization; dermatitides such as seborrheic
dermatitis and solar dermatitis; keratosis such as seborrheic
keratosis, senile keratosis, actinic keratosis. photo-induced
keratosis, and keratosis follicularis; acne vulgaris; keloids and
prophylaxis against keloid formation; nevi; warts including
verruca, condyloma or condyloma acuminatum, and human papilloma
viral (HPV) infections such as venereal warts; leukoplakia; lichen
planus; and keratitis.
[0129] As described above, 3-epi vitamin D.sub.3 compounds of
formula I can be used to inhibit the hyperproliferation of
keratinocytes in treating diseases such as psoriasis by
administering an effective amount of these compounds to a subject
in need of treatment. The term "psoriasis" is intended to have its
medical meaning, namely, a disease which afflicts primarily the
skin and produces raised, thickened, scaling, nonscarring lesions.
The lesions are usually sharply demarcated erythematous papules
covered with overlapping shiny scales. The scales are typically
silvery or slightly opalescent. Involvement of the nails frequently
occurs resulting in pitting, separation of the nail, thickening and
discoloration. Psoriasis is sometimes associated with arthritis,
and it may be crippling. Hyperproliferation of keratinocytes is a
key feature of psoriatic epidermal hyperplasia along with epidermal
inflammation and reduced differentiation of keratinocytes. Multiple
mechanisms have been invoked to explain the keratinocyte
hyperproliferation that characterizes psoriasis. Disordered
cellular immunity has also been implicated in the pathogenesis of
psoriasis.
[0130] As shown in Example XIV, 3-epi vitamin D.sub.3 compounds are
potent inhibitors of keratinocyte proliferation. Thus, providing
suitable agents for treatment of psoriasis in a subject, e.g. a
human.
[0131] Pharmaceutical compositions of 3-epi vitamin D.sub.3
compounds can be delivered or administered topically or by
transdermal patches for treating dermal psoriasis. Alternatively,
oral administration is used. Additionally, the compositions can be
delivered parenterally, especially for treatment of arthritis, such
as psoriatic arthritis, and for direct injection of skin lesions.
Parenteral therapy is typically intra-dermal, intra-articular,
intramuscular or intravenous. A preferred way to practice the
invention is to apply the vitamin D.sub.3 compound, in a cream or
oil based carrier, directly to the psoriatic lesions. Typically,
the concentration of vitamin D.sub.3 compound in a cream or oil is
1-2%. Alternatively, an aerosol can be used topically. These
compounds can also be orally administered.
[0132] In general, the route of administration is topical
(including administration to the eye, scalp, and mucous membranes),
oral, or parenteral. Topical administration is preferred in
treatment of skin lesions, including lesions of the scalp, lesions
of the cornea (keratitis), and lesions of mucous membranes where
such direct application is practical. Shampoo formulations are
sometimes advantageous for treating scalp lesions such as
seborrheic dermatitis and psoriasis of the scalp. Mouthwash and
oral paste formulations can be advantageous for mucous membrane
lesions, such as oral lesions and leukoplakia. Oral administration
is a preferred alternative for treatment of skin lesions and other
lesions discussed above where direct topical application is not as
practical, and it is a preferred route for other applications.
[0133] Intra-articular injection is a preferred alternative in the
case of treating one or only a few (such as 2-6) joints.
Additionally, the therapeutic compounds are injected directly into
lesions (intra-lesion administration) in appropriate cases.
Intra-dermal administration is an alternative for dermal lesions
such as those of psoriasis.
[0134] The amount of the pharmaceutical composition to be
administered varies depending upon the type of the disease of a
patient, the severity of the disease, the type of the active
3-epimeric form of vitamin D.sub.3, among others. For example, the
3-epi vitamin D3 compound of formula I can be administered
topically for treating hyperproliferative skin conditions at a dose
in the range of 1 to 1000 mg per gram of topical formulation.
[0135] Hormone Secretion
[0136] In yet another aspect, the present invention provides a
method for modulating hormone secretion of a vitamin D.sub.3
responsive cell, e.g., an endocrine cell, e.g., a parathyroid cell.
The language "hormone secretion" is art-recognized and includes
activities of vitamin D.sub.3 compounds that control the
transcription and processing responsible for secretion of a given
hormone e.g., parathyroid hormone (PTH) a vitamin D.sub.3
responsive cell (Bouillon, R. et al. (1995) Endocrine Reviews
16(2):235-237). The language "vitamin D.sub.3 responsive cells" as
used herein is intended to include endocrine cells which respond to
3-epi compounds of formula I by altering gene expression and/or
post-transcriptional processing secretion of a hormone. Exemplary
endocrine cells include parathyroid cells, among others.
[0137] The present method can be performed on cells in culture,
e.g. in vitro or ex vivo, or on cells present in an animal subject,
e.g., in vivo. 3-epi compounds of formula I can be initially tested
in vitro as discussed in Example XIV, which describes the
inhibition of PTH secretion in response to 3-epi vitamin D.sub.3
compounds in parathyroid cells in culture. Other systems that can
be used include the testing by prolactin secretion in rat pituitary
tumor cells, e.g., GH4Cl cell line (Wark J. D. and Tashjian Jr. A.
H. (1982) Endocrinology 111:1755-1757; Wark J. D. and Tashjian Jr.
A. H. (1983) J. Biol. Chem. 258:2118-2121; Wark J. D. and Gurtler
V. (1986) Biochem. J. 233:513-518) and TRH secretion in GH4C1
cells. Alternatively, the effects of 3-epi vitamin D.sub.3
compounds can be characterized in vivo using animals models as
described in Nko M. et al. (1982) Miner Electrolyte Metab. 5:67-75;
Oberg F. et al. (1993) J. Immunol. 150:3487-3495; Bar-Shavit Z. et
al. (1986) Endocrinology 118:679-686; Testa U. et al. (1993) J.
Immunol. 150:2418-2430; Nakamaki T. et al. (1992) Anticancer Res.
12:1331-1337; Weinberg J. B. and Larrick J. W. (1987) Blood
70:994-1002; Chambaut-Gurin A. M. and Thomopoulos P. (1991) Eur.
Cytokine New. 2:355; Yoshida M. et al. (1992) Anticancer Res.
12:1947-1952; Momparler R. L. et al. (1993) Leukemia 7:17-20;
Eisman J. A. (1994) Kanis J A (eds) Bone and Mineral Research
2:45-76; Veyron P. et al. (1999) Transplant Immunol. 1:72-76; Gross
M. et al. (1986) J. Bone Miner Res. 1:457-467; Costa E. M. et al.
(1985) Endocrinology 117:2203-2210; Koga M. et al. (1988) Cancer
Res. 48:2734-2739; Franceschi R. T. et al. (1994) J. Cell Physiol.
123:401-409; Cross H. S. et al. (1993) Naunyn Schmiedebergs Arch.
Pharmacol. 347:105-110; Zhao X. and Feldman D. (1993) Endocrinology
132:1808-1814; Skowronski R. J. et al. (1993) Endocrinology
132:1952-1960; Henry H. L. and Norman A. W. (1975) Biochem.
Biophys. Res. Commun. 62:781-788; Wecksler W. R. et al. (1980)
Arch. Biochem. Biophys. 201:95-103; Brumbaugh P. F. et al. (1975)
Am. J. Physiol. 238:384-388; Oldham S. B. et al. (1979)
Endocrinology 104:248-254; Chertow B. S. et al. (1975) J. Clin
Invest. 56:668-678; Canterbury J. M. et al. (1978) J. Clin. Invest.
61:1375-1383; Quesad J. M. et al. (1992) J. Clin. Endocrinol.
Metab. 75:494-501.
[0138] In certain embodiments, the 3-epi vitamin D.sub.3 compounds
of the present invention can be used to inhibit parathyroid hormone
(PTH) processing, e.g., transcriptional, translational processing,
and/or secretion of a parathyroid cell as part of a therapeutic
protocol. Therapeutic methods using these compounds can be readily
applied to all diseases, involving direct or indirect effects of
PTH activity, e.g., primary or secondary responses. For example, it
is known in the art that PTH induces the formation of
1,25-dihydroxy vitamin D.sub.3 in the kidneys, which in turn in
increases calcium and phosphate absorption from the intestine that
causes hypercalcemia. Thus inhibition of PTH processing and/or
secretion would indirectly inhibit all of the responses mediated by
PTH in vivo. Accordingly, therapeutic applications for these
vitamin D.sub.3 compounds include treating diseases such as
secondary hyperparathyroidism of chronic renal failure (Slatopolsky
E. et al. (1990) Kidney Int. 38:S41-S47; Brown A. J. et al. (1989)
J. Clin. Invest. 84:728-732). Determination of therapeutically
affective amounts and dose regimen can be performed by the skilled
artisan using the data described in the art.
[0139] Calcium and Phosphate Homeostasis
[0140] The present invention also relates to a method of treating
in a subject a disorder characterized by deregulation of calcium
metabolism. This method comprises contacting a pathological or
non-pathological vitamin D.sub.3 responsive cell with an effective
amount of 3-epi vitamin D.sub.3 compound of formula I to thereby
directly or indirectly modulate calcium and phosphate homeostasis.
The term "homeostasis" is art-recognized to mean maintenance of
static, or constant, conditions in an internal environment. As used
herein, the term "calcium and phospate homeostasis" refers to the
careful balance of calcium and phosphate concentrations,
intracellularly and extracellularly, triggered by fluctuations in
the calcium and phosphate concentration in a cell, a tissue, an
organ or a system. Fluctuations in calcium levels that result from
direct or indirect responses to 3-epi vitamin D.sub.3 compounds of
formula I are intended to be included by these terms. Techniques
for detecting calcium fluctuation in vivo or in vitro are known in
the art.
[0141] Exemplary Ca.sup.++ homeostasis related assays include
assays that focus on the intestine where intestinal
.sup.45Ca.sup.2+ absorption is determined either 1) in vivo
(Hibberd K. A. and Norman A. W. (1969) Biochem. Pharmacol.
18:2347-2355; Hurwitz S. et al. (1967) J. Nutr. 91:319-323; Bickle
D. D. et al. (1984) Endocrinology 114:260-267), or 2) in vitro with
everted duodenal sacs (Schachter D. et al. (1961) Am. J. Physiol
200:1263-1271), or 3) on the genomic induction of
calbindin-D.sub.28k in the chick or of calbindin-D.sub.9k in the
rat (Thomasset M. et al. (1981) FEBS Lett. 127:13-16; Brehier A.
and Thomasset M. (1990) Endocrinology 127:580-587). The
bone-oriented assays include: 1) assessment of bone resorption as
determined via the release of Ca.sup.2+ from bone in vivo (in
animals fed a zero Ca.sup.2+ diet) (Hibberd K. A. and Norman A. W.
(1969) Biochem. Pharmacol. 18:2347-2355; Hurwitz S. et al. (1967)
J. Nutr. 91:319-323), or from bone explants in vitro (Bouillon R.
et al. (1992) J. Biol. Chem. 267:3044-3051), 2) measurement of
serum osteocalcin levels [osteocalcin is an osteoblast-specific
protein that after its synthesis is largely incorporated into the
bone matrix, but partially released into the circulation (or tissue
culture medium) and thus represents a good market of bone formation
or turnover] (Bouillon R. et al. (1992) Clin. Chem. 38:2055-2060),
or 3) bone ash content (Norman A. W. and Wong R. G. (1972) J. Nutr.
102:1709-1718). Only one kidney-oriented assay has been employed.
In this assay, urinary Ca.sup.2+ excretion is determined
(Hartenbower D. L. et al. (1977) Walter de Gruyter, Berlin pp
587-589); this assay is dependent upon elevations in the serum
Ca.sup.2+ level and may reflect bone Ca.sup.2+ mobilizing activity
more than renal effects. Finally, there is a "soft tissue
calcification" assay that has been employed to detect the
consequences of 1.alpha.,25(OH).sub.2D.sub.3 or analog-induced
severe hypercalcemia. In this assay a rat is administered an
intraperitoneal dose of .sup.45Ca.sup.2+, followed by seven daily
relative high doses of 1.alpha.,25(OH).sub.2D.sub.3 or the analog
of interest; in the event of onset of a severe hypercalcemia, soft
tissue calcification can be assessed by determination of the
.sup.45Ca.sup.2+ level. In all these assays, either 3-epi-vitamin
D.sub.3 compound or related analogs are administered to vitamin
D-sufficient or-deficient animals, as a single dose or chronically
(depending upon the assay protocol), at an appropriate time
interval before the end point of the assay is quantified.
[0142] In certain embodiments, 3-epi vitamin D.sub.3 compounds of
formula I can be used to modulate bone metabolism. The language
"bone metabolism" is intended to include direct or indirect effects
in the formation or degeneration of bone structures, e.g., bone
formation, bone resorption, etc., which may ultimately affect the
concentrations in serum of calcium and phosphate. This term is also
intended to include effects of 3-epimer vitamin D.sub.3 compounds
in bone cells, e.g. osteoclasts and osteoblasts, that may in turn
result in bone formation and degeneration. For example, it is known
in the art, that vitamin D.sub.3 compounds exert effects on the
bone forming cells, the osteoblasts through genomic and non-genomic
pathways (Walters M. R. et al. (1982) J. Biol. Chem. 257:7481-7484;
Jurutka P. W. et al. (1993) Biochemistry 32:8184-8192; Mellon W. S.
and DeLuca H. F. (1980) J. Biol. Chem. 255:4081-4086). Similarly,
vitamin D.sub.3 compounds are known in the art to support different
activities of bone resorbing osteoclasts such as the stimulation of
differentiation of monocytes and mononuclear phagocytes into
osteoclasts (Abe E. et al. (1988) J. Bone Miner Res. 3:635-645;
Takahashi N. et al. (1988) Endocrinology 123:1504-1510; Udagawa N.
et al. (1990) Proc. Natl. Acad. Sci. USA 87:7260-7264).
Accordingly, 3-epi vitamin D.sub.3 that modulate the production of
bone cells can influence bone formation and degeneration.
[0143] The present invention provides a method for modulating bone
cell metabolism by contacting a pathological or a non-pathological
bone cell with an effective amount of a vitamin D.sub.3 compound of
formula I to thereby modulate bone formation and degeneration. The
present method can be performed on cells in culture, e.g., in vitro
or ex vivo, or can be performed in cells present in an animal
subject, e.g., cells in vivo. Exemplary culture systems that can be
used include osteoblast cell lines, e.g., ROS 17/2.8 cell line,
monocytes, bone marrow culture system (Suda T. et al. (1990) Med.
Res. Rev. 7:333-366; Suda T. et al. (1992) J. Cell Biochem.
49:53-58) among others. Selected compounds can be further tested in
vivo, for example, animal models of osteopetrosis and in human
disease (Shapira F. (1993) Clin. Orthop. 294:34-44).
[0144] In a preferred embodiment, a method for treating
osteoporosis is provided, comprising administering to a subject a
pharmaceutical preparation of a vitamin D.sub.3 compound to thereby
ameliorate the condition relative to an untreated subject. The
rationale for utilizing vitamin D.sub.3 compounds in the treatment
of osteoporosis is supported by studies indicating a decrease in
serum concentration of 1.alpha.,25(OH).sub.2D.sub.3 in elderly
subjects (Lidor C. et al. (1993) Calcif. Tissue Int. 52:146-148).
In vivo studies using vitamin D.sub.3 compounds in animal models
and humans are described in Bouillon, et al. (1995) Endocrine
Reviews 16(2):229-231.
[0145] 3-epi forms of vitamin D.sub.3 compounds of formula I can be
tested in ovarectomized animals, e.g., dogs, rodents, to assess the
changes in bone mass and bone formation rates in both normal and
estrogen-deficient animals. Clinical trials can be conducted in
humans by attending clinicians to determine therapeutically
effective amounts of the 3-epi compounds in preventing and treating
osteoporosis.
[0146] 3-epi forms of vitamin D.sub.3 compounds of formula I can be
tested in ovarectomized animals, e.g., dogs, rodents, to assess the
changes in bone mass and bone formation rates in both normal and
estrogen-deficient animals. Clinical trials can be conducted in
humans by attending clinicians to determine therapeutically
effective amounts of the 3-epi compounds in preventing and treating
osteoporosis.
[0147] The 3-epi vitamin D3 compounds of formula I are useful in
the treatment of senile osteoporosis. These compounds may be useful
in treating osteomalacia, rickets, osteitis fibrosa cystica, renal
osteodystrophy, osteosclerosis, anti-convulsant treatment,
osteopenia, fibrogenesis-imperfecta ossium, secondary
hyperparathyrodism, hyperparathyroidism, cirrhosis, obstructive
jaundice, drug induced metabolism, medullary carcinoma, chronic
renal disease, hypophosphatemic VDRR, vitamin D-dependent rickets,
sarcoidosis, glucocorticoid antagonism, malabsorption syndrome,
steatorrhea, tropical sprue, idiopathic hypercalcemia and milk
fever.
[0148] It is understood by the ordinary skilled artisan that
epimerization of a vitamin D.sub.3 substrate into a 3-epi vitamin
D.sub.3 compound in a cell is indicative that such compound is
biologically active in such cell, and thus that it can be used in
treating conditions arising from aberrant activity of such cells.
For example, production of 3-epi vitamin D.sub.3 compounds in
keratinocytes, smooth muscle cells and bone cells is indicative
that such 3-epi vitamin D.sub.3 compounds are biologically active
in those cells and can be used in treating conditions such as
psoriasis, hypertension and osteoporosis, respectively.
[0149] This invention is further illustrated by the following
examples which in no way should be construed as being further
limiting. The contents of all cited references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Example I
[0150] Isolation and Identification of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 as a Major Metabolite of
1.alpha.,25(OH).sub.2D.sub.3 in Human Keratinocytes
[0151] Keratinocytes were prepared as described previously (Kim H.
J. et al. (1992) Journal of Cellular Physiology 151:579-587) from
human foreskins. First passage keratinocytes were seeded at
0.5.times.10.sup.6 into 75 cm.sup.3 flasks in keratinocytes growth
media (KGM, Clonetics, Inc.). The cells were fed every 2-3 days.
The cells were 70-80% confluent usually after 5 days. The media was
changed to include 1.alpha.,25(OH).sub.2D.sub.3 (1 uM), 1.5 mM CaCl
and 0.2% bovine serum albumin. At the end of 24 hr incubation
period in a 37.degree. C., 5% CO.sub.2 incubator, an equal volume
of methanol was added to the cultures. The cells were then scraped
from the flasks and all of the media and the cells were pooled and
lipid extraction was performed from both cells and media using the
Bligh and Dyer technique. The lipid extract was then subjected to
HPLC directly for the separation of the various
1.alpha.,25(OH).sub.2D.sub.3 metabolites using the following HPLC
conditions. Zorbax-SIL column eluted with 6% isopropanol in hexane
at a flow rate of 2 ml/min.
[0152] FIG. 4 shows the HPLC profile of the metabolites of
1.alpha.,25(OH).sub.2D.sub.3 produced in human keratinocytes. The
insert in this Figure shows the UV spectra of the various
metabolites as monitored by a photodiode array detector. As
indicated in FIG. 4, Peak 1 is the substrate,
1.alpha.,25(OH).sub.2D.sub.3; peaks 2-6 exhibited UV spectra
typical of natural metabolites of vitamin D cis-triene chromophore.
All of these metabolites comigrated with the known natural
metabolites of 1.alpha.,25(OH).sub.2D.sub.3 formed through C-24 and
C-23 oxidation pathways as shown in FIG. 1. Accordingly, peaks 1-6
correspond to 1.alpha.,25(OH).sub.2D.sub.3,
1.alpha.,25(OH).sub.2-24-oxo-D.sub.3, C-23 Alcohol,
1.alpha.,23(S),25(OH).sub.3D.sub.3, 1.alpha.,23(S),25(OH).s-
ub.3-24-oxo-D.sub.3, and 1.alpha.,24(R)25(OH).sub.3D.sub.3. Peak C
is a lipid contaminant.
[0153] For the first time, a less polar peak migrating just prior
to the 1.alpha.,25(OH).sub.2D.sub.3 peak was detected. The unknown
peak was labeled as peak M, which was later identified as
1.alpha.,25(OH).sub.2-3-- epi-D.sub.3. This peak exhibited a UV
spectrum identical to other vitamin D metabolites, indicating an
intact cis-triene cromophore. The surprising finding of a novel
metabolite peak with the natural metabolites of
1.alpha.,25(OH).sub.2D.sub.3 produced in various target tissues,
led to further characterization of this peak by mass spectrometry
after several HPLC purification steps.
[0154] The mass spectrum of the metabolite is shown in the upper
panel of FIG. 5. For comparison, the lower panel of FIG. 5 shows
the mass spectrum of synthetic 1.alpha.,25(OH).sub.2D.sub.3
standard. The mass spectrum indicates that the molecular ion (m/z
416) and the mass fragmentation pattern of the metabolite were
identical to the standard, 1.alpha.,25(OH).sub.2D.sub.3. This
indicates that the metabolite M produced in keratinocytes is an
isomer of 1.alpha.,25(OH).sub.2D.sub.3 and therefore it has to be
one of the 3 possible diastereomers shown in FIG. 2. The only way
to identify the stereochemistry of the hydroxyl groups at the C-3
positions was to use the HPLC technique. As a result both straight
phase and reverse phase HPLC systems were developed.
[0155] The straight phase HPLC system was performed using a
Zorbax-SIL column eluted with 6% isopropanol in hexane at a flow of
2 ml/min, and the reverse phase HPLC system was performed using a
Zorbax-ODS column eluted with 20% methanol in water at a flow of 1
ml/min. The retention times of the various diastereomers is shown
in the Table of FIG. 6. On the straight phase HPLC system,
metabolite M comigrated with 1.alpha.,25(OH).sub.2-3-epi-D.sub.3.
However, the standard 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 did not
resolve very well from 1.beta.,25OH).sub.2D.sub.3 on this HPLC
system. As a result, the reverse phase HPLC which gave a good
separation between 1.alpha.,25(OH).sub.2-3-e- pi-D.sub.3 and
1.beta.,25(OH).sub.2D.sub.3 was developed. The metabolite
comigrated on the reverse phase system with only
1.alpha.,25(OH).sub.2-3-- epi-D.sub.3. Thus, from the mass spectral
and the HPLC data, the metabolite M was identified as
1.alpha.,25(OH).sub.2-3-epi-D.sub.3.
Example II
[0156] Metabolism of 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in Human
Keratinocytes
[0157] To determine whether 1.alpha.,25(OH).sub.2-3-epi-D.sub.3
would be converted back into 1.alpha.,25(OH).sub.2D.sub.3,
incubation studies were performed using
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 as a starting substrate.
Resulting lipid extracts were analyzed using the following HPLC
conditions: Zorbax-SIL column eluted with 9% isopropanol in hexane
at a flow rate of 2 ml/min. The results in FIG. 7 show that
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 is not converted back into
1.alpha.,25(OH).sub.2-D.sub.3, as evidenced by the absence of a
peak in the elution position of 1.alpha.,25(OH).sub.2D.sub.3.
However, extensive metabolism of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 into more polar metabolites can
be detected, and all of the metabolites exhibited typical vitamin D
UV spectra. Initial structure identification of one of the
metabolites indicated that it is a 24-hydroxylate form of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3. This finding indicates that
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 also undergoes side chain
modifications. Thus, it appears that modification of the A-ring did
not prevent side chain metabolism.
[0158] FIG. 8A shows a detailed HPLC profile of the
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 metabolites produced in human
keratinocytes. UV spectra of the various metabolites as monitored
by a photodiode array detector are shown in the insert. As
indicated in the insert box, peak M has been identified as
1.alpha.,25(OH).sub.2-3-epi-D.s- ub.3; peaks M.sub.1-M.sub.7 are
unidentified metabolites of 1.alpha.,25(OH).sub.2-3-epi-D.sub.3.
Peak M.sub.8 has been identified as
1.alpha.,24(R),25(OH).sub.3-3-epi-D.sub.3. Peaks C.sub.1-C.sub.3
are contaminants.
[0159] FIG. 8B summarizes the metabolism of
1.alpha.,25(OH).sub.2-3-epi-D.- sub.3 in keratinocytes. As
depicted, both 1.alpha.,25(OH).sub.2 and its 3-epi form are capable
of undergoing side chain metabolism through C-24 and C-23 oxidation
pathways.
Example III
[0160] Further Characterization of the Metabolism of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in Human Keratinocytes
[0161] Extensive metabolism of 1.alpha.,25(OH).sub.2-3-epi-D.sub.3
in primary cultures of human keratinocytes is shown in FIGS. 7 and
8A. The isolation and structure identification of metabolites has
tentatively been identified as a 24-hydroxylated metabolite of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3. To further characterize these
metabolites, rat kidney perfusions with micromolar concentration of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 were performed for a period of
4 to 8 hours. The metabolites of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 were isolated from the kidney
perfusate and their structures were determined through mass
spectrometry and specific chemical reactions. In this regard, not
only the lipid soluble metabolites, but also the water soluble
metabolites were analyzed. Using the metabolites of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 as standards, possible
differences in metabolism between 1.alpha.,25(OH).sub.2D.sub.3 and
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in other tissues which have the
ability to produce 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 can be
identified.
Example IV
[0162] Formation of 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 under
Physiological Substrate Concentration
[0163] Since the isolation and identification of the
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 were performed using micromolar
concentrations of 1.alpha.,25(OH).sub.2D.sub.3, it was important to
determine whether the formation of this metabolite is produced
under physiological substrate concentrations. Accordingly, human
keratinocytes were isolated from adult breast skin and incubated
with tritiated 25(OH)D.sub.3. Lipid extracts of different time
points were analyzed by straight phase HPLC. FIG. 9 shows the
radioactive profiles of tritiated 25(OH)D.sub.3 metabolites. As
shown, tritiated 25(OH)D.sub.3 (retention time 11-12 min) is
converted into tritiated 1.alpha.,25(OH).sub.2D.sub.3 (retention
time 38-39 min) which reaches its maximum by 1-2 hr. and decreases
to very low levels at 4 hr. The less polar metabolite migrating
before 1.alpha.,25(OH).sub.2D.sub.3 was identified as tritiated
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 (retention time 35-36 min).
This metabolite peak appeared at 1 hr. incubation period and
increased to its maximum by 3 hr. This metabolite peak also
decreased by 4-6 hr. Along with the decrease in both
1.alpha.,25(OH).sub.2D.sub.3 and
1.alpha.,25(OH).sub.2-3-epi-D.sub.3, an increase in the water layer
tritium counts but also the more polar metabolite peaks was
detected, indicating further metabolism of both
1.alpha.,25(OH).sub.2D.sub.3 and
1.alpha.,25(OH).sub.2-3-epi-D.sub.3. Thus, this result shows that
the new pathway described in the present invention also operates at
physiological substrate concentrations.
Example V
[0164] Structural Characterization of New Metabolites
[0165] As shown by the preceding Examples, the side chain of both
1.alpha.,25(OH).sub.2D.sub.3 and its 3-epimer are the major targets
for metabolic modifications. Oxidation results in the formation of
hydroxylated or keto products. Subsequent degradation of the side
chain also produces carboxylic acids. These metabolites can be
isolated by HPLC and characterized by conventional electron
ionization (EI) MS using the direct insertion probe sample inlet.
Typically, quantities on the order of 1.0 ug can be used for this
purpose. As deemed necessary, derivatization of hydroxyl groups by
silylation may be utilized in order to enhance the classical
cleavage beta to the heteroatom and help improve the intensity of
structurally informative ions. The occurrence of suspect vicinal
hydroxyl group may be recognized by conversion to n-butyl boronate
derivatives or, if adequate sample is available, the performance of
periodate cleavage and analysis of the products by GC-MS. More
polar metabolites like carboxylic acids may have to be esterified
prior to EI/MS analysis or, alternatively, may be analyzed by
electrospray ionization (ESI) and collisionally induced
dissociation (CID). Earlier work (Yeung B., Thesis, Northeastern
University, June 1995) has reported that compounds such as
calcitroic acid are very amenable to analysis by positive ion
ESI-MS with detection capabilities extending into the picogram
range. The introduction of positive ion ESI-MS for the detection of
calcitroic acid allows the detection capabilities extend into the
picogram range.
Example VI
[0166] Trace Level Detection of Metabolites
[0167] Trace level detection of metabolites of vitamin D can be
performed using the Cookson Reagent,
4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), which is a powerful
dienophile known to react selectively with conjugated dienes. It
has been shown (Yeung B. et al. (1993) Journal of Chromatography
645:115-123; Vreeeken R. J. et al. (1993) Biol. Mass Specrom.
22:621-632), that the reagent reacts quantitatively at the
sub-nanogram level with the 19/10-5/6 diene system of vitamin D via
a Diels-Alder reaction. In earlier work, we have relied on the use
of vitamin D-PTAD derivatives in conjunction with tandem MS for the
recognition of vitamin D related molecules in complex biologically
derived mixtures. The protonated molecules of PTAD derivatives of
vitamin D have been shown to fragments by collision induced
dissociation (CID) to give a characteristic ion of mass 298, or in
the case of 1-hydroxylated compounds such as
1.alpha.,25(OH).sub.2D.sub.3, an ion of mass 314. Since the typical
vitamin D metabolites are modified on the side chain or some other
part of the molecule, operation of the triple quadruple MS system
in the parent ion scan mode can effectively "fish" out of the
mixture all vitamin D molecules while also identifying their
molecular masses. This approach has been reported in the past to
establish the presence of a 24-oxo metabolite of
1.alpha.,25(OH).sub.2-16-ene-D.sub.3 in the rat kidney perfusate of
the parent analog (Yeung B. et al. (1994) Biochem. Pharmacol.
49:1099-1110). All of the above methods are described in detail in
(Yeung B. et al. (994) Biochem. Pharmacol. 49:1099-1110).
Example VII
[0168] Metabolism of 1.alpha.,25(OH).sub.2)D.sub.3 into
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in Bovine Parathyroid Cells
[0169] Bovine parathyroid glands obtained from a local
slaughterhouse were digested with collagenase and the cells were
cultured as previously described (Brown A. J. et al. (1992)
Endocrinology 130:276-281), and seeded at a density of 80,000
cells/cm.sup.2. Cells were grown to confluence in six days in serum
free medium. Confluent cultures of parathyroid cells were then
incubated with 1 uM 1.alpha.,25(OH).sub.2D.su- b.3 in serum free
medium for 24 hours. Incubations were terminated with methanol, and
the samples were sent to our laboratory for HPLC analysis. The
lipid extract was analyzed using the straight phase HPLC system
(Zorbax-SIL column eluted with 6% isopropanol inhexane at 2 ml/min
flow rate). As can be seen in the HPLC chromatogram,
1.alpha.,25(OH).sub.2D.su- b.3 is metabolized into other side chain
modified metabolites through both C-24 and C-23 oxidation
pathways.
[0170] FIG. 10 shows the HPLC profile of the metabolites of
1.alpha.,25(OH).sub.2D.sub.3 produced in bovine parathyroid cells.
Peaks in the chromatogram have been identified as the indicated
compounds. The significant finding of this study was the
recognition of a less polar metabolite peak migrating in front of
the 1.alpha.,25(OH).sub.2D.sub.3 peak. As shown in the insert, the
identified peak exhibited the same UV spectral characteristics
similar to 1.alpha.,25(OH).sub.2D.sub.3, indicating that this peak
is indeed a vitamin D metabolite with an intact cis-triene
cromophore. This metabolite was then isolated individually and
further purified on three different HPLC systems. The purified
metabolite was identified as 1.alpha.,25(OH).sub.2-3-epi-D.sub.3
through mass spectrometry and its comigration with synthetic
standard 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 on both straight phase
and reverse phase HPLC systems. These experiments have demonstrated
that, like the human keratinocytes, bovine parathyroid cells also
have the ability to convert 1.alpha.,25(OH).sub.2D.sub.3 into
1.alpha.,25(OH).sub.2-3-epi-D.s- ub.3.
Example VIII
[0171] Tissue Specificity of 3-.beta.-hydroxy Epimerization of
1.alpha.,25(OH).sub.2D.sub.3
[0172] To address the tissue-specific nature of the
3-.beta.-hydroxy epimerization reaction, the metabolism of
1.alpha.,25(OH).sub.2D.sub.3 in human placental explants was
investigated. Previous studies using the human placental explant
model had shown 24(R),25(OH).sub.2D.sub.3 and
23(R),25(OH).sub.2D.sub.3 as the major metabolites of
25(OH).sub.2D.sub.3 and accordingly, isolated both
1.alpha.,24(R),25(OH).sub.3D.sub.3 and
1.alpha.,23(R),25(OH).sub.3D.sub.3 were isolated from placental
explants. However, it is interesting to note that an organ like the
placenta, which as multiple enzyme activities was unable to
epimerize the 3-hydroxy group of 1.alpha.,25(OH).sub.2D.sub.3 (FIG.
11).
[0173] FIG. 12 shows a comparison of the metabolism of
1.alpha.,25(OH).sub.2D.sub.3 in four different tissues. Primary
cultures of human keratinocytes were used as a control (FIG. 12,
panel A). The other three tissues tested included a cell line of
immortalized human keratinocytes (HACAT) (FIG. 12, panel B), a
commonly studied cancer cell line (human promyelocytic leukemic
cell line, HL-60) (FIG. 12, panel C), and perfused rat kidney (FIG.
12, Panel D). All of these studies were carried on for 24 hr. using
1 uM 1.alpha.,25(OH).sub.2D.sub.3 as the substrate and the lipid
extracts were analyzed using the same HPLC systems described for
FIG. 5. As can be seen in FIG. 12, the peak M, which is now
identified as 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 is formed in
keratinocytes and the HACAT cells, but not in HL-60 cells and the
rat kidney. The experiments certainly establish the fact that the
3-epimerization is not ubiquitous, like the side chain oxidation
pathways.
[0174] Thus, these results indicate that the formation of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in primary cultures of
keratinocytes, immortalized keratinocyte cell line (HACAT cells),
primary cultures of bovine parathyroid cells and rat aortic
vascular smooth muscle cells.
Example IX
[0175] Metabolism and Pharmacokinetic Studies of
1.alpha.,25(OH).sub.2D.su- b.3 in Bone Cells
[0176] Previous examples described in the present application show
that the 3-.beta.-hydroxy epimerization reaction is target
tissue-specific. As described above, there is evidence of the
formation of 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 tissues diverse as
keratinocytes, parathyroid cells and aortic vascular smooth muscle
cells. These findings prompted us to characterize classical target
tissues of 1.alpha.,25(OH).sub.2D.sub.3, i.e., bone, kidney and
intestine, in terms of the presence of a similar pathway. The
metabolism of 1.alpha.,25(OH).sub.2D.sub.3 in primary cultures of
human bone cells can be studied using the above described
conditions. (Siu-Caldera M. L. et al. (1995) Endocrinology
136:4195-4203).
[0177] FIG. 13 shows the presence of a less polar metabolite of
1.alpha.,25(OH).sub.2D3 in the rat osteosarcoma cell line UMR 106.
Upper panels show HPLC profiles of metabolites after 24 hours of
addition of the indicated concentrations of
1.alpha.,25(OH).sub.2D.sub.3 (1 mM-20 mM). Lower panels show HPLC
profiles of metabolites after 48 hours of addition of the indicated
concentrations of 1.alpha.,25(OH).sub.2D.sub.3 (1 mM-20 mM). The
upper panel to the left shows the identified peaks. The formation
of 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 was dose dependent at the
concentrations tested (1 mM-20 mM). Surprisingly,
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 is more stable than other
metabolites, as indicated by the persistently high concentrations
of 3-epi metabolites after 48 hour incubation compared to other
metabolites. As shown in the lower panels after 48 hours, a
persistent peak can be detected which corresponds to
1.alpha.,25(OH).sub.2-3-epi-D.sub.3.
[0178] To further address the enhanced stability of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in rat osteosarcoma cells
(UMR-106), HPLC profiles were determined after 24 hours (upper
panel) and 48 hours (lower panel) of 1.alpha.,25(OH).sub.2D.sub.3
addition (FIG. 14). Insert panel shows the UV spectra of the
various metabolites as monitored by photodiode array detector. The
peaks have been identified as shown in the insert box. Peak 1
corresponds to 1.alpha.,25(OH).sub.2D.sub.3; peak M corresponds to
1.alpha.,25(OH).sub.2-3-epi-D.sub.3. Other metabolites have been
identified as indicated in the insert box. At 48 hours, all of the
starting substrate 1.alpha.,25(OH).sub.2D.sub.3) was metabolized
and the concentrations of all of the intermediary metabolites
remained almost the same as in the 24 h incubation. Out of all of
the remaining intermediary metabolites, the concentration of
1.alpha.,25(OH).sub.2-3-ep- i-D.sub.3 was the highest. The
continued presence of the M peak relative to other metabolites
after 48 hours demonstrates the enhanced stability of the 3-epi
form of 1.alpha.,25(OH).sub.2D.sub.3.
[0179] FIG. 15 shows the formation of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in a human osteosarcoma cell
(U-2 OS) grown at two different cell densities. HPLC profiles were
determined at two different cell densities, 3.times.10.sup.7 cells
and 12.times.10.sup.7 as shown in the upper and lower panels of the
Figure, respectively. Increased conversion into 3-epi forms of
vitamin D.sub.3 was directly proportional to the concentration of
cells. Insert panel shows the UV spectra of the various metabolites
as monitored by photodiode array detector. The peaks have been
identified as shown in the insert box. As before, Peak 1
corresponds to 1.alpha.,25(OH).sub.2D.sub.3; Peak M corresponds to
1.alpha.,25(OH).sub.2-3-epi-D.sub.3. Other metabolites have been
identified as indicated in the insert box.
[0180] These studies show that human bone cells, like
keratinocytes, also have the ability to produce
1.alpha.,25(OH).sub.2-3-epi-D.sub.3. Production of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 has been shown in the human
osteosarcoma cell line (U-2 OS) and the rat osteosarcoma cell line
(UMR 106). These studies can be extended to primary cultures of
human bone cells, isolated from different age groups of
patients.
Example X
[0181] Metabolism of 1.alpha.,25(OH).sub.2D.sub.3 in Target Tissues
of 1.alpha.,25(OH).sub.2D.sub.3
[0182] The presence of 3-.beta.-hydroxy epimerization can be
characterized in other classical target tissues of
1.alpha.,25(OH).sub.2D.sub.3. For example, the production of 3-epi
metabolites of 1.alpha.,25(OH).sub.2D.su- b.3 can be characterized
in the intestine by using the human colon cancer cell line (Caco-2
cells), which is a common cell line that responds to
1.alpha.,25(OH).sub.2D.sub.3. This cell line has been shown to
metabolize 1.alpha.,25(OH).sub.2D.sub.3 via the C-24 oxidation
pathway (Tomon M. et al. (1990) Endocrinology 126:2868-2875). In
order to investigate the presence of the 3-.beta.-hydroxy
epimerization reaction in normal intestinal tissue, intestinal
homogenates of both rat and chick can be incubated with A-ring
labeled tritiated 1.alpha.,25(OH).sub.2D.sub.3, followed by
performance of a time course to investigate the production
1.alpha.,25(OH).sub.2-3-epi-D.sub.3.
[0183] Although the experiments described above show the lack of
3-.beta.-hydroxy epimerization in the perfused rat kidney, the
possibility of the kidney as a site for 3-.beta.-hydroxy
epimerization through the kidney perfusion studies cannot be
completely ruled out since only the metabolites that enter into the
perfusate from the kidney were analyzed. Thus, it is still possible
that 1.alpha.,25(OH).sub.2-3-epi-D.s- ub.3 is formed
intracellularly and does not enter the perfusate. Therefore,
metabolism studies can be carried out by incubating homogenates of
both the chick and rat kidney to investigate the formation of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 using A-ring labeled
1.alpha.,25(OH).sub.2D.sub.3. Most kidney perfusions can be
performed in rats (300 gm) treated on a regular rodent diet
sufficient in vitamin D, calcium and phosphorous. In brief, the
rats can be anesthetized with Nembutal and right renal artery can
be cannulated and the right kidney isolated from the rat. Isolated
kidneys can be perfused with oxygenated perfusate which contains 6%
bovine albumin in Krebs-Henseleit bicarbonate buffer. The kidneys
are usually perfused at a mean arterial pressure of 100 mm of Hg
and a good functioning kidney should maintain a constant pressure.
The details of the kidney perfusion system are known in the
art.
[0184] The presence of the 3-.beta.-hydroxy epimerization reaction
in non-classical target tissues of 1.alpha.,25(OH).sub.2D.sub.3 can
be characterized as follows. Even though the liver has been a site
for the metabolism of 1.alpha.,25(OH).sub.2D.sub.3 and its
excretion into the bile through conjugation with glucuronic acid
and the site for the excretion of calcitroic acid into bile, it has
been clearly established that the liver has no enzymatic ability to
produce side chain modified metabolites through C-24 and C-23
oxidation pathways. As the 3-.beta.-hydroxy epimerization reaction
plays an important role in bile acid metabolism, 3-.beta.-hydroxy
epimerization can act as a possible means of inactivation of
1.alpha.,25(OH).sub.2D.sub.3 by the liver. The formation of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in the homogenates of both rat
and chick liver can be tested by the methods described herein. The
homogenates can be incubated using A-ring labeled
1.alpha.,25(OH).sub.2D.sub.3. The study of the 3-.beta.-hydroxy
epimerization can also be carried out in a human hepatoma cell line
(Hep 3B). This cell line was used previously to study
25-hydroxylation of vitamin D.sub.3. This study can be performed by
incubating uM concentration of 1.alpha.,25(OH).sub.2D.sub.3 for a
period of 24 hr. The isolation of the putative metabolite can then
allow the structure identification.
[0185] Other non-classical target tissues that can be caracterized
include neoplastic tissues. Recently, there has been great interest
in evaluating several of the noncalcemic metabolites and synthetic
analogs, e.g., metabolites of 1.alpha.,25(OH).sub.2D.sub.3 and one
of its analogs, 1,25(OH).sub.2-16-ene D.sub.3 in terms of their
ability to suppress the growth of several breast and prostate
cancer cell lines. At present, there is very little information in
terms of the ability of these cancer cell lines to metabolize the
hormone 1.alpha.,25(OH).sub.2D.sub.3. As these cancerous tissues
possess vitamin D.sub.3 receptor and respond to the hormone, it can
be predicted that these tissues would be able to metabolize
1.alpha.,25(OH).sub.2D.sub.3 and its analogs through side chain
modifications using 24-hydroxylase as the key enzyme. Studies can
be performed by incubating the confluent cells with micromolar
concentration of 1.alpha.,25(OH).sub.2D.sub.3 for a period of 24
hours and the analysis will be carried on by HPLC.
Example XI
[0186] Metabolism of Synthetic Vitamin D.sub.3 Analogs in Bone
Cells
[0187] In order to address whether vitamin D.sub.3 analogs can be
converted to 3-epi forms, a number of analogs were tested in bone
cells under identical experimental conditions as described in
Example IX. At present, all of the vitamin D.sub.3 analogs tested
have rapidly metabolized into their less polar 3-epi metabolites.
Similar conversions have been obtained using bovine PTH cells (data
not shown). These results show that the above-described findings
for 1.alpha.,25(OH).sub.2-3-epi-D.- sub.3 can be extended to other
vitamin D.sub.3 analogs.
[0188] FIG. 16 shows the conversion of
1.alpha.,25(OH).sub.2-16-ene-D.sub.- 3 into its 3-epi form in rat
osteosarcoma cell (UMR-106). The inserts in both panel show the UV
spectra of the various metabolites as monitored by photodiode array
detector. The chemical structures of the analogs are also provided.
The upper panel of this Figure shows the HPLC profile of
1.alpha.,25(OH).sub.2D.sub.3. Peak 3 corresponds to
1.alpha.,25(OH).sub.2-3-epi-D.sub.3. The lower panel of FIG. 16
shows the HPLC profile of 1.alpha.,25(OH).sub.2-16-ene-D.sub.3
metabolites. Peak 1a corresponds to the 3-epi form of this
analog.
[0189] Similarly, FIG. 17 shows the metabolism of
1.alpha.,25(OH).sub.2-20- -epi-D.sub.3 and
1.alpha.,25(OH).sub.2-16-ene-20-epi-D.sub.3 in the rat osteosarcoma
cell (UMR-106). The upper panel of this Figure shows the HPLC
profile of 1.alpha.,25(OH).sub.2-20-epi-D.sub.3. Peak 4 corresponds
to the 3-epi form of this compound. The lower panel of FIG. 17
shows the HPLC profile of
1.alpha.,25(OH).sub.2-16-ene-20-epi-D.sub.3 metabolites with peak
2a corresponding to the 3-epi form of this analog. As before, the
inserts in both panel show the UV spectra of the various
metabolites as monitored by photodiode array detector. The chemical
structures of the analogs are also provided. FIG. 18 summarizes the
HPLC profiles of the analogs tested in rat osteosarcoma cells
(UMR-106), indicating for all of the compounds tested, a less polar
3-epi metabolites was detected. As indicated, the peaks in each
chromatogram have been identified as a 3-epi or its substrate. The
chemical structures of these compounds are shown on the right of
the Figure.
[0190] FIG. 19 shows the metabolism of
1.alpha.,25(OH).sub.2-16-ene-D.sub.- 3 and
1.alpha.,25(OH).sub.2-16-ene-23-yne-D.sub.3 in the rat osteosarcoma
cell (UMR-106). Peaks M16e and M23y represent the 3-epi forms of
1.alpha.,25(OH).sub.2-16-ene-D.sub.3 and
1.alpha.,25(OH).sub.2-16-ene-23-- yne-D.sub.3, respectively. S
peaks correspond to the substrate. Other metabolites of
1.alpha.,25(OH).sub.2-16-ene-D.sub.3 are also indicated. The insert
panels show the UV spectra of the various metabolites as monitored
by photodiode array detector. The chemical structures of these
compounds are shown on the right of the Figure.
[0191] In sum, these studies show that vitamin D.sub.3 analogs are
converted into 3-epi forms in bone cells as efficiently as
1.alpha.,25(OH).sub.2-D.sub.3. Thus, the above-described findings
for 1.alpha.,25(OH).sub.2-D.sub.3 can be extended to vitamin
D.sub.3 analogs.
Example XII
[0192] Metabolism of Synthetic Vitamin D.sub.3 Analogs in Human
Colon Adenocarcinoma-Derived Caco-2 Cells
[0193] To address whether the epimerization of vitamin D3 analogs
occurs in other tissues, the intestinal cell line Caco-2 was used
as a model system to investigate the metabolism of two synthetic
analogs of 1,25(OH).sub.2D.sub.3 in intestinal epithelial cells.
Subconfluent (6 days after seeding) and confluent (14 days after
seeding) cells were incubated with 1 .mu.M
1,25(OH).sub.2-16-ene-D.sub.3 or
1,25(OH).sub.2-16-ene-23-yne-D.sub.3, respectively, for 48 hours.
HPLC analysis of lipid extracts revealed that subconfluent cells
when incubated with 1,25(OH).sub.2-16-ene-D.sub.3, produced large
amounts of two metabolites more polar than
1,25(OH).sub.2D.sub.3.
[0194] In confluent cells these two metabolites could not be
detected (data not shown). However, another single peak appeared,
which eluted before 1,25(OH).sub.2D.sub.3. Two peaks produced by
subconfluent cells have been indentified as as
1,24,25(OH).sub.3-16-ene-D.sub.3 and 1,25 (OH)2-24-oxo-16-ene-D3,
and the single peak found in confluent cells only was identified as
the 3-epi form of 1,25(OH)2-16-ene-D3 (data not shown). When Caco-2
cells were incubated with 1,25(OH)2-16-ene-23-yne-D3, no
metabolites could be detected in subconfluent cells (data not
shown).
[0195] 3-epi forms of 1,25(OH).sub.2-16-ene-23-yne-D.sub.3 were
identified in confluent cells (data not shown). It is suggested
that the triple bond between C23 and C24 inhibits further
metabolism via modification of the side chain while this has no
influence on structural changes of the A-ring. Under all growth
conditions, 1,25(OH).sub.2-16-ene-23-yne-D.sub.3 was metabolized
considerably slower than 1,25(OH).sub.2D3 or
1,25(OH).sub.2-16-ene-D3 (data not shown).
[0196] These data indicate that rapidly dividing Caco-2 cells
metabolize vitamin D3 compounds preferentially through side chain
oxidation, while 3-OH epimerization is a hallmark of vitamin D
metabolism in postconfluent differentiating cells.
Example XIII
[0197] Metabolism of 1.alpha.,25(OH).sub.2-16-ene-D.sub.3 Human
Keratinocytes
[0198] Previous reports have shown that
1.alpha.,25(OH).sub.2-16-ene-D.sub- .3 is metabolized differently
from 1.alpha.,25(OH).sub.2D.sub.3 in rat kidney and human leukemic
cells (J. Steroid Biochem. Molec. Biol. 59:405-412, 1996). The
16-ene modification hinders the further metabolism of the
intermediary metabolite, 1.alpha.,25(OH).sub.2-16-ene-24-oxo-D.sub-
.3, thus allows this metabolite to accumulate. This metabolite, in
turn, exhibited equipotent effect as its parent compound in
modulating proliferating and differentiation of human leukemic
cells (data not shown). In another study
1.alpha.,25(OH).sub.2-16-ene-D.sub.3 was shown to be the most
potent of the analogs tested in inhibiting proliferation and
inducing differentiation of keratinocytes (J. Invest. Dermatol.
101:713-718, 1993).
[0199] Therefore, the pattern of metabolism of this analog in human
keratinocytes was investigated. Comparative metabolism studies
indicated that human keratinocytes also metabolize
1.alpha.,25(OH).sub.2-16-ene-D.s- ub.3 differently, and the
differences were similar to the ones previously observed in kidney
and leukemic cells. The significant finding was the accumulation of
1.alpha.,25(OH).sub.2-16-ene-24-oxo-D.sub.3 which exceeded the
amounts of remaining unmetabolized substrate. Biological activity
studies indicated that, as in leukemic cells, both
1.alpha.,25(OH).sub.2-16-ene-D.sub.3 and its 24-oxo metabolite were
equally potent at inhibiting growth of keratinocytes. The
biological activites of these compounds were more potent than
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 (data not shown).
[0200] In addition, the identification of a less polar metabolite
of 1.alpha.,25(OH).sub.2-16-ene-D.sub.3 and
1.alpha.,25(OH).sub.2D.sub.3 was detected in human keratinocytes
that is not observed in kidney nor leukemic cells (Example VIII).
The less polar metabolite of 1.alpha.,25(OH).sub.2D.sub.3 has been
identified as 1.alpha.,25(OH).sub.2-3-epi-D.sub.3, thus it is
possible that the less polar metabolite of
1.alpha.,25(OH).sub.2-16-ene-D.sub.3 is the putative
1.alpha.,25(OH).sub.2-16-ene-3-epi-D.sub.3.
Example XIV
[0201] Biological Activities of 1.alpha.,25(OH).sub.2D.sub.3 and
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in Keratinocytes and Bovine
Parathyroid Cells
[0202] To address the effect of 3-epi vitamin D.sub.3 compounds on
keratinocyte growth, keratinocyte cultures were established at
25,000 cells/per well on day 0 in KGM. On day 1, the cultures were
refed with KGM supplemented with 1.5 mM Ca.sup.++ containing either
vehicle or different concentrations of
1.alpha.,25(OH).sub.2D.sub.3. The cells were allowed to grow for 4
more days with one refeeding on day 3. Number of cells per well was
determined on day 4. The experiment was repeated at least 3 times
with similar results, and these results are shown in FIG. 20, panel
A.
[0203] To determine the effect of 3-epi vitamin D.sub.3 compounds
on parathyroid hormone secretion, bovine parathyroid cells were
prepared as described in the metabolism studies. These cells were
grown for four days in serum-free media. The cells were then
treated for 3 days with either 1.alpha.,25(OH).sub.2D.sub.3 or its
3-epimer at different concentrations. Steady state PTH seretion was
determined by washing the cells 3 times with Dulbecco's PBS and
then placing them in serum free media for 3 hours. The media was
collected, centrifuged at 4.degree. C. and analyzed from PTH using
CH9 antibody as described previously (Brown A. J. et al. (1992)
Endocrinology 130:276-281). The cell monolayers were dissolved in
0.1 N NaOH and assayed for protein by method of Bradford using a
kit from Biorad Laboratories. PTH secretion is expressed as
picograms PTH per milligram cell protein (FIG. 20, panel B).
Example XV
[0204] Improved Biological Properties of
1.alpha.,25(OH).sub.2D.sub.3 and
1.alpha.,25(OH).sub.2-3-epi-D.sub.3
[0205] As shown in Example XIV, 1.alpha.,25(OH).sub.2-3-epi-D.sub.3
shows significant biological activity as evidenced by its ability
to suppress keratinocyte growth and inhibit PTH secretion. Tissue
incubation studies shown above indicate that in prolonged
incubations, the concentration of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 is significantly higher when
compared to the unmetabolized 1.alpha.,25(OH).sub.2D.sub.3
substrate (Example VIII, FIGS. 13 and 14). These data indicate that
3-epi forms of vitamin D.sub.3 are more stable in vivo compared to
their isomeric counterparts.
[0206] The enhanced stability of 3-epi metabolites in vivo is
further evidenced in Table 1 below, which shows an eightfold
increase in the binding of 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 to
plasma vitamin D binding protein (DBP) compared to
1.alpha.,25(OH).sub.2D.sub.3 (A. W. Norman et al. J. Biol. Chem.
268 (27): 20022-20030). These studies measure the relative ability
of 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 to compete with
1.alpha.,25(OH).sub.2D.sub.3 for binding to DBP in routine ligand
binding studies. The elevated levels of 3-epi binding indicate that
3-epi forms bind to DBP more avidly than their isomeric
counterparts. Such enhanced binding may in turn enhance the
stability of 3-epi forms in the plasma, and thus may explain the
prolonged presence of 3-epi metabolites in vivo.
[0207] Ligand binding assays are well known in the art. In brief,
increasing concentrations of a nonradioactive, test analog are
incubated with a fixed saturating amounts of
[3H]1.alpha.,25(OH).sub.2D.sub.3; the reciprocal of the percentage
of maximal binding of [3H]1.alpha.,25(OH).sub.2D.sub.3 can then be
calculated and plotted as a function of the relative concentration
of the test analog. Such plots give linear curves characteristic
for each test analog, the slopes of which are equal to the analog's
competitive index (Wecksler, W. R. and Norman, A. W. (1980) Methods
of Enzymol. 67:494-500). The competitive index value for each
analog is then normalized to a standard curve obtained with
radioactive 1.alpha.,25(OH).sub.2D.sub.3 as the competing steroid
and placed on a linear scale of relative competitive index (RCI),
where the RCI of 1.alpha.,25(OH).sub.2D.sub.3 is by definition
100.
[0208] The reduced hypercalcemic activity of the 3-epi vitamin
D.sub.3 compounds is evidenced by the reduced level of bone calcium
mobilization (BCM) and intestinal calcium absorption (ICA) induced
by 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 as illustrated in Table 1
below and described in Norman, A. W. et al. (1993) J. Biol. Chem.
268(27):20022-20029. 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 showed a
dramatic reduction in BCM activity (1.5 compared to 100), and ICA
activity (2.8 compared to 100) compared to
1.alpha.,25(OH).sub.2-D.sub.3. Thus, 3-epi forms of vitamin D.sub.3
show remarkably reduced hypercalcemic activity in vivo.
[0209] Compounds exhibiting reduced hypercalcemic activity can be
tested in vivo or in vitro using methods known in the art and
reviewed by Boullion, R. et al. (1995) Endocrinology Reviews 16(2):
200-257. For example, the serum calcium levels following
administration of a vitamin D.sub.3 compound can be tested by
routine experimentation (Lemire, J. M. (1994) Endocrinology
135(6):2818-2821). Briefly, 3-epi vitamin D.sub.3 compounds can be
administered intramuscularly to vitamin D.sub.3-deficient subjects,
e.g., rodents, e.g. mouse, or avian species, e.g. chick. At
appropriate time intervals, serum calcium levels and extent of
calcium uptake can be used to determine the level of BCM and ICA
induced by the tested vitamin D.sub.3 compound, as illustrated in
Table 1 below and described in Norman, A. W. et al. (1993) J. Biol.
Chem. 268(27):20022-20029. Compounds which upon addition fail to
increase the concentration of calcium in the blood serum, thus
showing decreased BCM and ICA responses compared to their isomeric
counterparts, are considered to have reduced hypercalcemic
activity.
[0210] The 3-epi metabolite retains most of
1.alpha.,25(OH).sub.2D.sub.3 non-genomic activity as measured by
transcaltachia (Table 1 below; VDR binding 25%; Transcaltachia
80%). In addition, as shown in Example XIV, this metabolite has
significant activities in suppressing keratinocyte growth and PTH
secretion. Although 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 show
reduced genomic activities than its isomeric counterpart, other
3-epi analogs of vitamin D.sub.3 can retain genomic activities as
described below in Example XVII.
1TABLE 1 COMPARATIVE BIOLOGICAL ACTIVITIES BETWEEN
1.alpha.,25(OH).sub.2D.sub.3 AND 1.alpha.,25(OH).sub.2-3-epi-D.sub-
.3 1.alpha.,25(OH).sub.2D.sub.3 1.alpha.,25(OH).sub.2-3-epi-D.sub.-
3 (1.alpha.,3.beta.) (1.alpha.,3.beta.) Binding to DBP (RCI) 100
800 Binding to VDR* (RCI) 100 24 Intestinal Calcium Transport 100
2.8 Bone Calcium Mobilization 100 1.5 Osteocalcin Synthesis MG-63
100 17 Transcaltachia 100 80 previously published by A.W. Norman et
al. (J Biol. Chem. 268 (27): 20022-20030).
[0211] For the experiments described hereinafter, HPLC system to
separate various metabolites of 1.alpha.,25(OH).sub.2D.sub.3 and
its analogs have been described in detail in the following
publications: Biochemistry 28:1763-1769, (1989) (Baran D. T. and
Sorensen A. M. (1994) Proc. Soc. Exp. Biol. Med. 207:175-178);
Endocrinology 136:4195-4203, (1995) (Siu-Caldera M. L. et al.
(1995) Endocrinology 136:4195-4203). Unlabeled versions of four
diastereomers of 1.alpha.,25(OH).sub.2D.sub.3 and
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 can be converted into their
corresponding 1-.sup.3H-labeled compounds using the synthetic
scheme illustrated in FIG. 21.
Example XVI
[0212] Pharmacokinetic Studies Between 1.alpha.,25(OH).sub.2D.sub.3
and 1.alpha.,25(OH).sub.2-3-epi-D.sub.3.
[0213] Tissue incubation studies shown above indicate that
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 is one of the major metabolites
of 1.alpha.,25(OH).sub.2D.sub.3, and in prolonged incubations, the
concentration of 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 is
significantly higher when compared to the unmetabolized
1.alpha.,25(OH).sub.2D.sub.3 substrate. This phenomenon can be
clearly seen in the HPLC chromatogram shown in FIGS. 13 and 14, in
which 1.alpha.,25(OH).sub.2D.sub.3 metabolism was studied in bovine
parathyroid cells incubated with 1 .mu.M
1.alpha.,25(OH).sub.2D.sub.3 for a period of 24 hours. From these
observations, it appeared that the rate of further metabolism of
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 is significantly slower than
its parent 1.alpha.,25(OH).sub.2D.sub.3. It is possible that the
affinity of 24-hydroxylase for 1.alpha.,25(OH).sub.2-3-epi-D.sub.3
may be less when compared to the affinity for
1.alpha.,25(OH).sub.2D.sub.3 . Significant accumulation of this
metabolite can be of importance, as it has been shown to possess
significant biological activity in the tissues where it is formed.
For example, significant biological activity was demonstrated in
keratinocytes and bovine parathyroid cells (FIG. 20).
[0214] Kidney perfusion studies can be performed comparing the
rates of disappearance of 1.alpha.,25(OH).sub.2D.sub.3 and
1.alpha.,25(OH).sub.2-3- -epi-D.sub.3 by perfusing kidneys
independently with each compound for a period of four hours with
varying substrate concentrations. With the availability of the
A-ring labeled tracer of both substrates, pharmacokinetic studies
can be performed at very low substrate levels and can be used to
calculate disappearance rates for these two epimers. Also, at the
same time, information can also be gained about the selective
accumulation of some of the intermediary metabolites which may have
significant biological activities. As the rat kidney is not a site
of 1.alpha.,25(OH).sub.2-3-epi-D.sub.3 formation, pharmacokinetic
studies can also be repeated using the cell line (HACAT cells) in
which the epimer itself is produced. In this regard, the HACAT cell
culture system can be used, and the rates of metabolism of
1.alpha.,25(OH).sub.2D.sub.3 and
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 in these cells can be compared
by incubating the cells with different concentrations of
1.alpha.,25(OH).sub.2D.sub.3 and
1.alpha.,25(OH).sub.2-3-epi-D.sub.3 for different time periods.
Example XVII
[0215] Transcription Activities of 3-epi Analogs of Vitamin
D.sub.3
[0216] To test whether 3-epi analogs mediated transcription
activation of vitamin D.sub.3 nuclear receptors (VD.sub.3R),
ROS-17/2.8 cells were transfected with a construct containing the
hormone gene as a reporter gene under the control of the
osteocalcin vitamin D receptor response element (VDRE). The
preparation of constructs, culture and transfection of ROS-17/2.8
cells were carried out following standard protocols. In this assay,
expression of the hormone gene is indicative of induction of
VD.sub.3R by the vitamin D3 compounds tested. Transfected
ROS-17/2.8 cells were contacted with
1.alpha.,25(OH).sub.2-16-ene-23-yne-3-epi D.sub.3 and its isomeric
counterpart, and the transcriptional activity induced was
monitored. The transcriptional activity of
1.alpha.,25(OH).sub.2D.sub.3 was also measured for comparison. FIG.
23 shows that both 1.alpha.,25(OH).sub.2-16-ene-23-yne-3-epi
D.sub.3 and its isomeric counterpart induce transcriptional
activity VD.sub.3R in a dose dependent manner. Although the
activation induced by 1.alpha.,25(OH).sub.2-16-ene-23-yne-3-epi
D.sub.3 is slightly decreased in relation to its isomeric
counterpart, this 3-epi analog is as active as
1.alpha.,25(OH).sub.2D.sub.3 in mediating transcriptional activity.
These results indicate that 3-epi analogs of vitamin D3 can retain
similar genomic activities as their isomeric counterparts.
Example XVIII
[0217] Synthesis of
[3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-Bis[[(1,1-dimethyle-
thyl)-dimethylsilyl]oxy]-2-methylenecyclohexylidene]ethyl]diphenylphosphin-
e Oxide (Compound of Formula II)
[0218] 3-epi vitamin D.sub.3 compounds of formula I were prepared
by a convergent synthesis which involved reacting an anion
corresponding to
[3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-Bis[[(1,1-dimethylethyl)-dimethylsilyl-
]oxy]-2-methylenecyclohexylidene]ethyl]diphenylphosphine oxide
(referred to herein as the compound of formula II), with a starting
compound (for example, a compound represented by the formula IIIb-i
of FIG. 23B), followed by removal of the protecting silyl groups
with tetra-n-butylammonium fluoride in tetrahydrofuran at room
temperature.
[0219] The synthesis of the compound of formula II was performed by
a sequence of reactions starting from the known compound of formula
IV summarized as Exp. 1-11 of FIG. 23A and described in detail
below as Reactions 1-11. The various starting materials and
intermediates produced during the synthesis are identified as
compounds IV-XIV of FIG. 23A.
[0220] Reaction 1: Synthesis of
2S-[2.alpha.,3.alpha.,(R*)]]-7-Hydroxy-4-m-
ethylene-1-oxaspiro[2.5]octane-2-methanol Acetate (Compound of
Formula V)
[0221] As shown in the formula scheme depicted below, compound of
formula IV was converted into the compound of formula V. The
compound of formula V was obtained by removal of the protecting
silyl group with tetra-n-butylammonium fluoride in tetrahydrofuran
as a solvent. 6
[0222] The synthesis was performed as follows. To 220 ml. of 1M
tetrabutylammonium fluoride in a 1-L r.b. flask provided with
magnetic stirrer and argon atmosphere was added rapidly 70.5 g.
(0.216 mol.) of
[2S-[2.alpha.,3.alpha.,(R*)]]-7-[[(1,1-dimethylethyl)-dimethylsilyl]oxy]--
4-methylene-1-oxa-spiro[2.5]octane-2-methanol acetate. Complete
transfer of the oily silyl ether from its container was effected by
rinsing with a total of 150 ml. of tetrahydrofuran. After 22 hr.
starting material still remained, so an additional 35 ml. of 1M
tetrabutylammonium fluoride was added. After 3 hr. the reaction
mixture was poured into 2 L. of water and extracted 4 times with 1
L. of ethyl acetate. The organic phases were washed in a
counter-current manner twice with 500 ml. of water. The combined
organic phases were evaporated under reduced pressure and the
residue chromatographed on 527 g. of silica gel (eluted with 4:1
hexanes-ethyl acetate to ethyl acetate). Recovered starting
material 10.94 g. (16%) was followed by 34.46 g. (75% yield) of
[2S-[2.alpha.,3.alpha.,(R*)]]-7-hydroxy-4-methylene-1-oxaspiro[2.5]octane-
-2-methanol acetate as a colorless oil: [.alpha.]S(25,D)
+2.7.degree. (c=1.2, CHCl.sub.3); MS 213.2 (M+1); .sup.1H NMR
(CDCl.sub.3) .delta. 2.10 (s, 3H), 2.16 (dd, J=13, 2 Hz, 1H), 2.36
(br d, J=12 Hz, 1H), 2.54 (br t, J=2 Hz, 1H), 3.20 (t, J=5 Hz, 1H),
3.98 (dd, J=12, 6 Hz, 1H), 4.17 (dd, J=12, 5 Hz, 1H). 4.34 (br s,
1H), 4.92 (s, 1H), 4.95 (s, 1H); Anal. Calcd for
C.sub.11H.sub.16O.sub.4: C, 62.25; H, 7.60. Found C, 62.10; H,
7.54.
[0223] Reaction 2: Synthesis of [2S-[2 .alpha.,3
.alpha.,(S*)]]-4-Methylen-
e-7-(4-nitrobenzoyloxy)-1-oxaspiro[2.5]-octane-2-methanol Acetate
(Compound of Formula VI)
[0224] As shown in the formula scheme depicted below, the compound
of formula VI was obtained by reaction of the compound of formula V
with p-nitrobenzoic acid, triphenylphosphine and diethyl
azodicarboxylate in toluene as a solvent at 8-10.degree. C.
temperature. 7
[0225] The synthesis was performed as follows. To a suspension of
33.87 g. (203 mmol.) of p-nitrobenzoic acid and 53.24 g. (203
mmol.) of triphenylphosphine in 350 ml. of toluene contained in a
2-L. r. b. flask provided with argon atmosphere and magnetic
stirrer was added at 10.degree. C. (ice/water bath) 35.35 g. (203
mmol.) of diethyl azodicarboxylate rapidly dropwise. The
temperature rose to 20.degree. C. After five minutes the
temperature dropped back to 10.degree. C. and a solution of 35.84
g. (169 mmol.) of [2S-[2a,3a,(R*)]]-7-hydroxy-4-methyle-
ne-1-oxaspiro[2.5]octane-2-methanol acetate in 200 ml of toluene
was added over about 5 minutes. Precipitation commenced in about 15
minutes. After 3 hours at 8-10.degree. C. the yellow reaction
mixture was poured into 250 ml. of 10% sodium bicarbonate. The
suspension was extracted successively with 700 ml. (leaving the
undissolved solid with the aqueous phase), 500 ml. (all solids
dissolved), and 250 ml. of ethyl acetate. The combined organic
phases were dried (Na.sub.2SO.sub.4), filtered, and evaporated. The
residue was triturated with 200 ml. of ether and the insoluble
white solid (EtOOC--NH--NH--COOEt and triphenylphospine oxide) was
removed by filtration. The filtercake was washed with ether and the
white solid checked by nmr and tlc to verify the absence of
product. The ether filtrate was evaporated and the residue (89 g.)
was chromatographed (medium pressure) on three 0.5 m..times.55 mm.
columns in series (silica gel G-60) using 4:1 hexanes-ethyl acetate
as elution solvent. Twenty-four 250 ml. fractions were collected.
Fractions 14-18 (33.64 g.) were pure product. The overlapping
fractions (1-13 and 19-24) amounted to 15.41 g and were
rechromatographed in the same solvent system using only two 0.5
m..times.55 mm. columns in series connected to an automatic
fraction collector. A total of 200 25-ml. fractions was eluted.
Fractions 61-81 contained 3.62 g. (9% yield) of colorless oil (The
nmr was compatible with the corresponding .DELTA..sup.2,3
elimination product.). Fractions 121-170 contained 9.47 g. of
product. The combined total from both chromatograms amounted to
43.11 g. (71% yield) of [2S-[2.alpha.,3.alpha.,-
(S*)]]-4-methylene-7-(4-nitrobenzoyloxy)-1-oxaspiro[2.5]-octane-2-methanol
acetate as an off-white solid. An analytical sample, obtained by
recrystallization from ethyl acetate/hexanes, had m.p.
81-82.degree. C.; [.alpha.]S(25,D) -43.4.linevert split.(c0.98,
EtOH); HRMS (M+H), observed 362.1248, theor. 362.1241; .sup.1H NMR
(CDCl.sub.3) .delta. 1.70 (m, 1H), 1.92 (br d, J=12 Hz, 1H), 2.11
(s, 3H), 2.25 (m, 3H), 2.60 (d, J=13 Hz, 1H), 3.25 (t, J=6 Hz, 1H),
4.02 (dd, J=12, 6 Hz, 1H), 4.10 (dd, J=12, 5 Hz, 1H), 5.03 (s, 1H),
5.05 (s, 1H), 5.26 (m, 1H), 8.20 (d, J=9 Hz, 2H), 8.30 (d, J=9 Hz,
2H); Anal. Calcd for C.sub.18H.sub.19NO.sub.7: C, 59.83; H, 5.30;
N, 3.88. Found: C, 59.27; H, 5.22; N, 3.82.
[0226] Reaction 3: Synthesis of
[2S-[2.alpha.,3.alpha.,(S*)]]-7-Hydroxy-4--
methylene-1-oxaspiro[2.5]octane-2-methanol Acetate (Compound of
Formula VII)
[0227] As shown in the formula scheme depicted below, the compound
of formula VII was obtained by hydrolysis of the compound of
formula VI with sodium hydroxide in a mixture of dioxane and water
as a solvent. 8
[0228] The synthesis was performed as follows. To a vigorously
stirred solution of 44.16 g. (122 mmol.) of [2S-[2 .alpha.,3
.alpha.,(S*)]]-4-methylene-7-(4-nitrobenzoyloxy)-1-oxaspiro[2.5]-octane-2-
-methanol acetate in 500 ml. of dioxane and 50 ml. of water
contained in a 3-L 3-necked r.b. flask fitted with thermometer and
argon inlet and immersed in an ice-water bath was added at
8.degree. C. dropwise over 40 min. a solution of 4.88 g (122 mmol.)
of sodium hydroxide in 50 ml. of water. After 1 hr. an additional
0.49 g (12 mmol.) of sodium hydroxide pellets was added. Stirring
at 8.degree. C. was continued for an other hour, and then the
reaction solution was poured into a 2-L separatory funnel; 100 ml.
of brine and 25 ml. of 1N sodium bicarbonate were added followed by
extraction 4 times with 1-L portions of ethyl acetate (the diol is
water soluble). The organic phases were dried (Na.sub.2SO.sub.4),
filtered, and the solvents removed on a rotary evaporator. The
extracts amounted to 1) 35.28 g. 2) 1.49 g. 3) 0.52 g. 4) 0.14 g.
The combined extracts were flash chromatographed on 155 g. of
silica gel G60 in 4:1 hexanes-ethyl acetate. Elution with 2:1
hexanes-ethyl acetate gave a mixture of di- and mono-esters. Ethyl
acetate eluted 5.24 g. (25% yield) of crystalline diol,
[2S-[2.alpha.,3.alpha.,(S*)]]-7-hydroxy-4-methylene--
1-oxaspiro[2.5]octane-2-methanol. An analytical sample was
recrystallized from acetonitrile to give a white solid, m.p.
90.5-91.5; [.alpha.]S(25,D) -2.3.degree. (c0.99, CHCl.sub.3); MS
(M+H) 171.1; .sup.1H NMR (CDCl.sub.3) .delta. 1.42 (m, 1H), 1.74
(dd, J=12, 2 Hz, 1H), 2.01 (m, 3H), 2.45 (m, 1H), 3.12 (m, 3H),
3.54 (s, 2H), 3.91 (br t, 1H), 4.90 (s, 1H), 4.92 (s, 1H); Anal.
Calcd for C.sub.9H.sub.14O.sub.3: C, 63.51; H, 8.29; N,3.88. Found:
C, 63.66; H, 8.42.
[0229] The above ester mixture was chromatographed on two 0.5
m..times.55 mm. columns (silica gel G-60) in series (medium
pressure) connected to an automatic fraction collector. A total of
283 25-ml. fractions were collected. The earlier fractions
consisted of mostly starting material 11.93 g (27% yield) and minor
amounts of di p-nitrobenzoate (1.67 g., 3% yield), primary
p-nitrobenzoate 3-OH (0.71 g., 2% yield), 3-p-nitrobenzoate primary
OH (3.61 g., 9% yield). Fractions 255-282 contained 8.03 g. (31%)
of the desired product, [2S-[2.alpha.,3.alpha.,(S-
*)]]-7-hydroxy-4-methylene-1-oxaspiro[2.5]octane-2-methanol
acetate. as an oil: [.alpha.]S(25,D) -7.9.linevert split.(c1.18,
CHCl.sub.3); MS (M.sup.+), 212.1; .sup.1H NMR (CDCl.sub.3) .delta.
1.45 (br dd, 1H), 1.66 (s, 1H), 1.74 (m, 1H), 2.04 (m, 3H), 2.09
(s, 3H), 2.50 (m, 1H), 3.16 (t, J=5 Hz, 1H), 3.95 (m, 2H), 4.10
(dd, J=12, 5 Hz, 1H), 4.97 (s, 2H), 5.26 (m, 1H); Anal. Calcd for
C.sub.11H.sub.16O.sub.4: C, 62.25; H, 7.60. Found: C, 61.50; H,
7.62.
[0230] Reaction 4: Synthesis of
[2.alpha.,3.alpha.,(S*)]]-7-[[(1,1-dimethy-
lethyl)-dimethylsilyl]oxy]-4-methylene-1-oxaspiro[2.5]octane-2-methanol
Acetate (Compound of the Formula VIII)
[0231] As shown in the formula scheme depicted below, the compound
of formula VIII was obtained from the compound of formula VII by
reaction with t-butyldimethylsilyl chloride in the presence of
imidazole in dimethylformamide as a solvent. 9
[0232] The synthesis was performed as follows. To a magnetically
stirred solution of 20.22 g. (95.2 mmol.) of
[2S-[2.alpha.,3.alpha.,(S*)]]-7-hydr-
oxy-4-methylene-1-oxaspiro[2.5]octane-2-methanol acetate and 10.2
g. (150 mmol.) of imidazole in 50 ml. of dimethyl-formamide under
an argon atmosphere was added 20.0 g. (133 mmol.) of
t-butyldimethylsilyl chloride. The reaction was allowed to stir
overnight (14 hr.) and 10 ml. of methanol was added. After 1 hr.
the reaction solution was poured into a separatory funnel
containing 500 ml. of water. Extraction with 2.times.250 ml. of
hexanes followed by countercurrent backwashes with 2.times.250 ml.
of water, afforded after drying (Na.sub.2SO.sub.4), filtration, and
evaporation under reduced pressure, 32.30 g. of an oil.
Chromatography on two 0.5 m..times.55 mm. columns (silica gel G-60)
in series (medium pressure) using 95:5 hexanes-ethyl acetate gave
29.19 g. (94% yield) of
[2.alpha.,3.alpha.,(S*)]]-7-[[(1,1-dimethylethyl)-dimethyl-
silyl]oxy]-4-methylene-1-oxaspiro[2.5]octane-2-methanol acetate as
a colorless oil: [.alpha.]S(25,D) -7.9.degree. (c1.02 CHCl.sub.3);
MS 326.1; H NMR (CDCl.sub.3) .delta. 0.066 (s, 3H), 0.072 (s, 3H),
0.88 (s, 9H), 1.42 (br m, 1H), 1.59 (br d, 1H), 1.97 (m, 3H), 2.09
(s, 3H), 2.45 (m, 1H), 3.12 (t, J=5 Hz, 1H), 3.88 (m, 2H), 4.12
(dd, J=12,5 Hz, 1H), 4.92 (s, 1H), 4.94 (s, 1H); Anal. Calcd for
C.sub.17H.sub.30O.sub.4Si: C, 62.54; H, 9.26; Si, 8.60. Found C,
62.69; H, 9.32; Si, 8.32.
[0233] Reaction 5: Synthesis of [2.alpha.,3.alpha.,(R*
,S*)]]-7-[[(1,1-dimethylethyl)-dimethylsilyl]oxy]-5-hydroxy-4-methylene-1-
-oxaspiro[2.5]octane-2-methanol Acetate (Compound of Formula
IX)
[0234] As shown in the formula scheme depicted below, the compound
of formula IX is obtained from the compound of formula VIII by
oxidation with selenium dioxide and tert.-butylhydroperoxide in
dioxane as a solvent at the temperature of 88.degree. C. 10
[0235] The synthesis was performed as follows. To a 2-L flask
provided with mechanical stirrer, argon atmosphere, and thermometer
containing a solution of 29.10 g. (89.1 mmol.) of
[2.alpha.,3.alpha.,(S*)]]-7-[[(1,1-d-
imethylethyl)-dimethylsilyl]oxy]-4-methylene-1-oxaspiro
[2.5]octane-2-methanol acetate in 1 L. of dioxane was added 11.1 g.
(100 mmol.) of pulverized selenium dioxide followed by 40 ml. of 3
M (120 mmol.) tert.-butylhydroperoxide in 2,2,4-trimethylpentane.
The stirred suspension was heated on the steam bath (88.degree. C.
pot temperature) for 7 hr. (the color gradually changed to dirty
red), and then allowed to cool overnight. The reaction suspension
was poured into a separatory funnel containing a solution of 69 g.
(0.5 mol.) of potassium carbonate and 25.2 g. (0.2 mol.) of sodium
sulfite in 300 ml. of water. Extraction with 3 L of 2:1
hexanes-ethyl acetate and 1L of 1:1 hexanes-ethyl acetate followed
by successive countercurrent washes with 500 ml. of water, 200 ml.
of 1 N sodium carbonate (red color), and 200 ml. of brine gave,
after drying the combined organic phases (Na.sub.2SO.sub.4),
filtration and evaporation under reduced pressure, 32.68 g. of
reddish oil. Successive flash chromatography on 90 g. and then 200
g. of silica gel G60 using hexanes to 9:1 hexanes-ethyl acetate
separated the starting material 2.91 g. (10%) and 3:1 hexanes-ethyl
acetate gave the hydroxylated products 21.57 g. Chromatography on
three 0.5 m..times.55 mm. columns in series (medium pressure) using
20:1 hexanes-isopropanol gave 4.74 g. (14% yield) of minor isomer,
[2.alpha.,3.alpha., (S*,S*)]]-7-[[(1,1-dimethylethyl)-di-
methylsilyl]oxy]-5-hydroxy-4-methylene-1-oxaspiro[2.5]octane-2-methanol
acetate as an oil: [.alpha.]S(25,D) -54.9.degree. (c1.06, EtOH); MS
342 (M+); .sup.1H NMR (CDCl.sub.3) .delta. 0.07 (s, 3H), 0.08 (s,
3H), 0.88 (s, 9H), 1.99 (t, J=12 Hz, 1H), 2.09 (s, 3H), 2.23 (br d,
J=14 Hz, 1H), 3.20 (t, J=6 Hz, 1H), 3.90 (dd, J=12, 6 Hz, 1H), 4.24
(dd, J=12, 5 Hz, 1H), 4.33 (m, 1H), 4.52 (or t, 1H), 5.08 (s, 1H),
5.16 (s, 1H); Anal. Calcd for C.sub.17H.sub.30O.sub.5Si: C, 59.62;
H, 8.83; Si, 8.20. Found C, 59.30; H, 8.68; Si, 7.97. and 13.50 g.
(45% yield) of major isomer,
[2.alpha.,3.alpha.,(R*,S*)]]-7-[[(1,1-dimethylethyl)-dimethylsilyl]oxy]-5-
-hydroxy-4-methylene-1-oxaspiro[2.5]octane-2-methanol acetate, as a
white solid, which, on recrystallization from hexanes, had m.p.
65-66.degree. [.alpha.]S(25,D) -2.5.linevert split.(c1.02, EtOH);
MS 341 (M-1); .sup.1H NMR (CDCl.sub.3) .delta. 0.07 (s, 3H), 0.08
(s, 3H), 0.88 (s, 9H), 1.50 (q, J=11 Hz, 1H), 1.60 (br d, J=12 Hz,
1H), 2.02 (t, J=12 Hz, 1H), 2.09 (s, 3H), 3.11 (t, J=5 Hz, 1H),
3.93 (m, 2H), 4.09 (m, 2H), 5.12 (s, 1H), 5.28 (s, 3H); Anal. Calcd
for C.sub.17H.sub.30O.sub.5Si: C, 59.62; H, 8.83; Si, 8.20. Found
C, 59.68; H, 8.83; Si, 8.18.
[0236] Reaction 6: Synthesis of
[2.alpha.,3.alpha.,(R*,S*)]]-5,7-bis-[[(1,-
1-dimethylethyl)-dimethylsilyl]oxy]-4-methylene-1-oxaspiro[2.5]octane-2-me-
thanol Acetate (Compound of Formula X)
[0237] As shown in the formula scheme depicted below, the compound
of formula X is obtained from the compound of formula IX by
reaction with t-butyldimethylsilyl chloride in the presence of
imidazole in dimethyl-formamide as a solvent at room temperature.
11
[0238] The synthesis was performed as follows. To a magnetically
stirred solution of 14.40 g. (42.0 mmol.) of
[2.alpha.,3.alpha.,(R*,S*)]]-7-[[(1,-
1-dimethylethyl)-dimethylsilyl]oxy]-5-hydroxy-4-methylene-1-oxaspiro[2.5]o-
ctane-2-methanol acetate and 5.1 g. (75 mmol.) of imidazole in 60
ml. of dimethylformamide under an argon atmosphere was added 7.54
g. (50 mmol.) of t-butyldimethylsilyl chloride. The reaction was
allowed to stir over the weekend (3 days) and then 5 ml. of water
was added. After 30 min. the reaction solution was poured into a
separatory funnel containing 400 ml. of water. Extraction with
2.times.400 ml. of 95:5 hexanes-ethyl acetate which were backwashed
in a countercurrent manner with 2.times.300 ml. of water and then
combined, dried (Na.sub.2SO.sub.4), filtered and evaporated under
reduced pressure gave 19.13 g. of amberish oil. Chromatography on a
0.5 m..times.55 mm. column (silica gel G-60) in (medium pressure)
using 10:1 hexanes-ethyl acetate gave 17.57 g. (92% yield) of
[2.alpha.,3.alpha.,(R*,S*)]]-5,7-bis-[[(1,1-dimethylethyl)-dime-
thylsilyl]-oxy]-4-methylene-1-oxaspiro [2.5]octane-2-methanol
acetate as a colorless oil: [.alpha.]S(25,D) -4.5.linevert
split.(c1.07, EtOH); MS 457 (M+1); .sup.1H NMR (CDCl.sub.3) .delta.
0.06 (s, 12H), 0.88 (s, 9H), 0.92 (s, 9H), 2.01 (t, J=12 Hz, 1H),
2.09 (s, 3H). 3.09 (t, J=6 Hz, 1H), 3.85 (m, 1H), 4.00 (m, 3H),
5.06 (s, 1H), 5.29 (s, 1H); Anal. Calcd for
C.sub.23H.sub.44O.sub.5Si.sub.2: C, 60.48; H, 9.71; Si, 12.38.
Found C, 60.49; H, 9.68; Si, 12.25.
[0239] Reaction 7: Synthesis of
[3R-(3.beta.,5.beta.)]-2-[3,5-Bis[[(1,1-di-
methylethyl)-dimethylsilyl]oxy]-2-methylenecyclohexylidene]-ethanol
Acetate (Cis/Trans Mixture) (Compounds of Formulae XI and XII)
[0240] As shown in the formula scheme depicted below, the compounds
of formulae XI and XII as a mixture were obtained from the compound
of formula X by reaction with anhydrous tungsten hexachloride and
n-butyl lithium in the mixture of tetrahydrofuran and hexane as
solvent at the temperature below -60.degree. C. 12
[0241] The synthesis was performed as follows. A 3-L. 3-neck flask
fitted with argon inlet, mechanical stirrer, and thermometer was
charged with 330 ml. of tetrahydofuran and cooled to -70.degree. C.
in a dry ice-acetone bath. Portionwise addition of 46.23 g. (116
mmol.) of anhydrous tunsten hexachloride was carried out while
maintaining the temperature below -60.degree. C., and then rapid
dropwise addition of 225 ml. of 1.6M (360 mmol.) butyllithium in
hexanes keeping the temperature below -45.degree. C. (ca 5 min.).
After 5 min. the dry ice/acetone bath was replaced with a icewater
bath. The exotherm caused the temperature to reach 12.degree. C.
before dropping to 5.degree. C. Color changes from blue to khaki to
reddish black occurred. After 30 min. at 5.degree. C., a solution
of 17.3 g. (37.9 mmol.) of [2.alpha.,3.alpha.,(R*,S*)]]-5,7-bis--
[[(1,1-dimethylethyl)-dimethyl-silyl]oxy]-4-methylene-1-oxaspiro[2.5]octan-
e-2-methanol acetate in 50 ml. of tetrahydrofuran was added rapidly
dropwise over 3 min. After 4.5 hr. the reaction mixture was diluted
with 750 ml. of hexanes and rapidly filtered through 400 g. of tlc
grade silica gel 60G packed (dry) tightly under vacuum in a 2-L.
sintered glass funnel. The filter cake was washed with 2.times.1L.
of 20:1 hexanes-ethyl acetate. The combined filtrates were
evaporated under reduced pressure at 25.degree. bath temperature to
give 17.47 g. of oil. Flash chromatography on 157 g. of silica gel
G60 gave 16.93 g. of oil. Chromatography on three 0.5 m..times.55
mm. columns in series (medium pressure) and elution with 100:1
dichlormethane-ethyl acetate afforded 14.20 g. (85% yield) of
[3R-(3.beta.,5.beta.)]-2-[3,5-Bis[[(1,1-dimethylethyl)-dimethylsilyl]oxy]-
-2-methylene-cyclohexylidene]-ethanol acetate as a 62/38 mixture of
trans/cis isomers.
[0242] Reaction 8: Synthesis of
[3R-(1Z,3.alpha.,5.alpha.)]-2-[3,5-Bis[[(1-
,1-dimethylethyl)-dimethylsilyl]oxy]-2-methylenecyclohexylidene]-ethanol
Acetate (Compound of Formula XII)
[0243] As shown in the formula scheme depicted below, the compound
of formula XI in the mixture with the compound of formula XII was
converted to compound of formula XII by irradiation with a 450-watt
Hanovia lamp with uranium core filter in the presence of fluorene
in tert.-butyl methyl ether as a solvent at room temperature.
13
[0244] The conversion was performed as follows. A solution of 14.13
g. of
3R-(3.alpha.,5.alpha.)]-2-[3,5-Bis[[(1,1-dimethylethyl)-dimethylsilyl]oxy-
]-2-methylenecyclohexylidene]-ethanol acetate (62/38 mixture of
trans/cis isomers) and 15 g. of fluorene in 500 mol. of tert.-butyl
methyl ether was irradiated with a 450-watt Hanovia lamp with
uranium core filter for 80 hr. After evaporation of the solvent
under reduced pressure, the residue was chromatographed on three
0.5 m..times.55 mm. columns in series (medium pressure) in 4 passes
(overlapping fractions rechromatographed) using 100:1
dichlormethaneethyl acetate to realize 12.88 g. (91% yield) of pure
cis isomer [3R-(1Z,3.alpha.,5.alpha.)]-2-[3,-
5-Bis[[(1,1-dimethylethyl)-dimethylsilyl]oxy]-2-methylenecyclohexylidene]--
ethanol acetate as a colorless oil: .sup.1H NMR (CDCl.sub.3)
.delta. 0.06 (m, 12H), 0.88 (s, 9H), 0.92 (s, 9H), 2.06 (s, 3H),
3.70 (m, 1H), 3.95 (m, 1H), 4.53 (m, 1H), 4.69 (m, 1H), 4.80 (s,
1H), 5.34 (s,1H), 5.46 (m, 1H).
[0245] Reaction 9:
[3R-(1Z,3,5.alpha.)]-2-[3,5-Bis[[(1,1-dimethylethyl)-di-
methylsilyl]oxy]2-methylenecyclohexylidene]-ethanol (Compound of
Formula XIII)
[0246] As shown in the formula scheme depicted below, the compound
of formula XIII was obtained from the compound of formula XII by
hydrolysis with sodium hydroxide in ethanol as a solvent. 14
[0247] The synthesis was performed as follows. To a magnetically
stirred solution of 12.88 g. (29.22 mmol.) of
[3R-(1Z,3.alpha.,5.alpha.)]-2-[3,5--
Bis[[(1,1-dimethylethyl)-dimethylsilyl]oxy]-2-methylenecyclohexyl-idene]-e-
thanol acetate in 100 ml. of 2B ethanol under an argon atmosphere
was added 2.0 g. (50 mmol.) of sodium hydroxide pellets. After 40
min. the reaction solution was poured into a separatory funnel
containing 400 ml. of brine. Extraction with 400 ml. of 5:3
hexanes-ethyl acetate followed by 250 ml. of ethyl acetate with a
countercurrent backwash with 200 ml. of brine gave after combining
the organic phases, drying (Na.sub.2SO.sub.4), filtration, and
evaporation under reduced pressure gave 11.51 g. of an oil.
Chromatography on two 0.5 m..times.55 mm. columns in series (medium
pressure) using 8:1 hexanes-ethyl acetate gave 10.77 g. (92% yield)
of [3R-(1Z,3.alpha.,5.alpha.)]-2-[3,5-Bis[[(1,1-dime-
thylethyl)-dimethylsilyl]oxy]-2-methylenecyclohexylidene]-ethanol
as a colorless oil: .sup.1H NMR (CDCl.sub.3) .delta. 0.07 (s, 6H),
0.08 (s, 3H), 0.09 (s, 3H), 0.88 (s, 9H), 0.93 (s, 9H), 1.56 (m,
2H), 2.17 (m, 2H), 2.43 (m, 1H), 3.73 (m, 1H), 3.96 (m, 1H), 4.14
(m, 1H), 4.29 (dd, J=12, 8 Hz, 1H), 4.77 (s, 1H), 5.33 (s, 1H),
5.55 (m, 1H).
[0248] Reaction 10: Synthesis of [1
S-(1a,3a,5Z)]-[[5-(2-chloroethylidene)-
-4-methylene-1,3cylohexanediyl]bis(oxy)]bis[(1,1-dimethylethyl)dimethyl]si-
lane (Compound of Formula XIV)
[0249] As shown in the formula scheme depicted below, the compound
of formula XIV was obtained from the compound of formula XIII by
reaction with N-chlorosuccinimide in dichlomethane as a solvent at
0.degree. C. temperature. 15
[0250] The synthesis was performed as follows. To a stirred
solution of 7.80 g. (58.4 mmol.) of N-chlorosuccinimide in 210 ml.
of dichloromethane under an argon atmosphere cooled to 2.degree. C.
in an iceacetone bath was added dropwise over 2 min. 4.5 ml. (61
mmol.) of dimethylsulfide. A white precipitate formed. After 30
min. at 0.degree. C., the bath was replaced with dry ice-acetone
and the pot temperature adjusted to -20.degree. C. by partial
immersion of the reaction flask. A solution of 10.62 g. (26.6
mmol.) of [3R-(1Z,3.alpha.,5.alpha.)]-2-[3,5-Bis[[(1,1-dim-
ethylethyl)-dimethylsilyl]-oxy]-2-methylenecyclohexylidene]-ethanol
in 30 ml. of dichloromethane was added dropwise over 10 min. After
30 min. the bath was replaced by icewater and stirring was
continued for 2 hr. at 0.degree. C. to 5.degree. C. at which time
the reaction mixture was transfered to a separatory funnel
containing 200 ml. of water. Extraction 2.times.300 ml. with
hexanes followed by backwashes with 2.times.250 ml. of water in a
countercurrent manner afforded, after combining the organic phases,
drying (Na.sub.2SO.sub.4), fil-tration, and evaporation under
reduced pressure, 11.3 g. Flash chromatography on 62 g. of silica
gel G60 followed by chromatography on a 0.5 m..times.55 mm. column
(medium pressure) using 95:5 hexanes-ethyl acetate gave 10.53 g.
(95% yield) of
[1S-(1.alpha.,3.alpha.,5Z)]-[[5-(2-chloro-ethylidene)-4-methylene-1,3-cyl-
ohexanediyl]bis(oxy)]bis[(1,1-dimethyl-ethyl)dimethyl]silane as a
colorless oil: .sup.1H NMR (CDCl.sub.3) .delta. 0.06 (m, 12H), 0.88
(s, 9H), 0.93 (s, 9H), 2.15 (m, 1H), 2.42 (m, 1H), 3.70 (m, 1H),
3.92 (m, 1H), 4.12 (m, 2H), 4.95 (s, 1H), 5.37 (s, 1H), 5.55 (m,
1H).
[0251] Reaction 11: Synthesis of
[3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-Bis[[(-
1,1-dimethylethyl)-dimethylsilyl]oxy]-2-methylenecyclohexylidene]ethyl]dip-
henylphosphine Oxide (Compound of Formula II)
[0252] As shown in the formula scheme depicted below, the compound
of formula II was obtained from compound of formula XIV in a
reaction with potassium diphenylphosphide in anhydrous
tetrahydrofuran as a solvent at -65.degree. C. temperature,
followed by oxidation with 30% hydrogen peroxide in a
water-dichloromethane mixture as a solvent at room temperature.
16
[0253] The synthesis was performed as follows. A 1-L. 3-neck flask
fitted with argon inlet, thermometer, and mechanical stirrer was
charged with a solution of 10.05 g. (24.1 mmol.) of
[1S-(1a,3a,5Z)]-[[5-(2-chloroethylid-
ene)-4-methylene-1,3-cylohexanediyl]bis(oxy)]bis[(1,1-dimethylethyl)dimeth-
yl]silane in 125 ml. of freshly distilled anhydrous
tetra-hydrofuran and cooled in a dry iceacetone bath to -65.degree.
C. Addition of 0.5 M potassium diphenyphosphide in tetrahydrofuran
during 30 min. until a red color persisted required 47 ml. After
stirring for 1 hr. at -65.degree. C. 10 ml. of water was added, and
the cooling bath removed. The reaction decolorized. Then 200 ml. of
dichloromethane was added rapidly followed by 60 ml. of a solution
containing 6.6 ml. of 30% hydrogen peroxide. After 1.4 hr 6.6 g. of
sodium sulfite followed by 100 ml of brine and 200 ml. of
dichloromethane was added. The phases were separated and the
aqueous phase was washed with 200 ml of dichloromethane. The
organic phases were backwashed in a countercurrent manner with 200
ml of brine. The combined organic phases were dried
(Na.sub.2SO.sub.4), filtered, and evaporated under reduced pressure
to give 16.08 g. of oil, which on medium pressure chromatography a
0.5 m.times.55 mm column (silica gel G-60), gave 12.22 g. (87%
yield) of [3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-bi-
s[[(1,1-dimethylethyl)-dimethylsilyl]oxy]-2-methylenecyclohexylidene]ethyl-
]diphenylphosphine oxide as a white solid. An analtyical sample,
recrystallized from acetonitrile, had m.p. 97-99.degree.;
[.alpha.]S(25,D) +5.8.degree.; MS 583(M+1); .sup.1H NMR
(CDCl.sub.3) .delta. 0.03 (s, 9H), 0.04 (s, 3H), 0.86 (s, 9H), 0.91
(s, 9H), 1.44 (q, J=11 Hz, 1H), 2.05 (br m, 2H), 2.37 (br d, 1H),
3.15 (br m, 1H), 3.35 (m, 2H), 3.52 (m, 1H), 4.75 (s, 1H), 5.27 (s,
1H), 5.48 (m, 1H), 7.5 (m, 6H), 7.7 (m, 4H); Anal. Calcd for
C.sub.33H.sub.51O.sub.3PSi.sub.2: C, 68.00; H, 8.82; P, 5.31; Si,
9.64. Found C, 67.46; H,8.74; P, 5.38; Si, 9.63.
Example XIX
[0254] Synthesis of 3-epi-1.alpha., 25-dihydroxycholecalciferol
[0255] Synthesis of 3-epi-1.alpha.-hydroxycholecalciferol was
carried out by reacting the compound of formula II with the
compound of formula IIIb followed by removal of the protecting
silyl groups with tetra-n-butylammonium fluoride in tetrahydrofuram
at room temperature as depicted in the formula scheme below. The
compound of formula IIIb is disclosed by Baggiolini E. et al.,
Journal Organic Chemistry 51, 3098-3108 (1986). 17
[0256] The synthesis was performed as follows. To a stirred, cold
(-78.degree. C.) solution of 582.9 mg (1.0 mL) of the reagent
[3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-bis[[(1,1-dimethyl)-dimethylsilyl]oxy]-
-2-methylenecyclohexylidene]ethyl]diphen-ylphosphine oxide in 5.0
mL of anhydrous THF was added 0.63 mL (1.0 mmol) of a 1.6 M
solution of n-butyllithium in hexanes. The resultant deep red
solution was stirred at -78.degree. C. for 15 minutes and treated
with 176 mg (0.5 mmol) of [1R-[1.alpha.(R*), 3a.alpha.,
7a.beta.]]-1-1,5-dimethyl-5-[(trimethylsily-
l)oxy]-hexyl]-octahydro-7a-methyl-4H-inden-4-one in 3.0 mL of THF.
The mixture was stirred at -78.degree. C. for 2.5 hours, allowed to
warm to room temperature, stirred for an additional 30 minutes and
quenched with 10 mL of a 1:1 mixture of 2.0 M Rochelle salt
solution and 2.0 N KHCO.sub.3 solution. After 15 minutes, the
mixture was poured into 75 mL of ethyl acetate and 50 mL of a 1:1
mixture of 2.0 M Rochelle salt solution and 2.0 N KHCO.sub.3
solution. The organic phase was separated and the aqueous phase was
re-extracted with 3.times.60 mL of ethyl acetate. The combined
organic extracts were dried (Na.sub.2SO.sub.4) and evaporated to
give a semisolid, which was purified by flash chromatography on 50
grams of silica gel (40-65 .mu.m mesh; 3.5 cm diameter column) with
8% ethyl acetate in hexanes taking 15-mL fractions. Fractions 4 and
5 were combined and evaporated to give 321 mg of a colorless gum.
The latter was dissolved in 5.0 mL of THF and treated with 4.0 mL
(4.0 mmol) of a 1.0 M solution of tetra-n-butylammonium fluoride in
THF. The mixture was stirred at room temperature for 17.0 hours,
diluted with 10 mL of water and, after 15 minutes, poured into a
mixture of 75 mL of ethyl acetate and 60 mL of 10% brine. The
organic phase was separated and the aqueous phase was re-extracted
with 3.times.70 mL of ethyl acetate. The combined organic extracts
were washed with 4.times.100 mL of water, dried (MgSO.sub.4) and
evaporated to give 197 mg of a gum, which was purified by flash
chromatography on 45 grams of silica gel (40-65 .mu.m mesh; 3.5 cm
diameter column) with ethyl acetate as eluent taking 15-mL
fractions. Fractions 12-21 were combined and evaporated to give 174
mg of a semisolid, which was dissolved in 15 mL of ethyl acetate.
The solution was filtered through a 0.4 .mu.m filter and the
filtrate was evaporated to give a solid. Crystallization from 7.0
mL of anhydrous methyl formate at -1.degree. C. overnight to give
160 mg of the title compound as colorless crystals, mp 135-136
.linevert split.C; [.alpha.]S(25,D) -43.88.degree.(MeOH, c=0.72);
UV (MeOH) 263 (.epsilon.=17,170), 218 (.epsilon.=12,405 shoulder),
213 (.epsilon.=13191) nm; IR (CHCl.sub.3) 3607, 3519 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3) .delta. 0.54 (3 H, s), 0.93 (3 H, d, J=6.8
Hz), 1.05 (1 H, m), 1.21 (6 H, s), 1.22-1.60 (19 H, m), 1.70 (2 H,
m), 1.90 (1 H, m), 1.96-2.09 (4 H, m), 2.17 (1 H, d, J=5 HzOH),
2.43 (1 H, m), 2.56 (1 H, d, J=12 Hz), 2.58 (1 H, d, J=12.8 Hz),
4.08 (1 H, br s) 4.30 (1 H, br s), 5.00 (1 H, s), 5.30 (1 H, s),
6.03 (1 H, d, J=12 Hz), 6.43 (1 H, d, J=12 Hz); MS (EI) Calcd. for
C.sub.27H.sub.44O.sub.3: m/z 416.3290. Found: m/z 416.3286. The
stereo-structure of the title compound was confirmed by a single
crystal X-ray analysis.
Example XX
[0257] Synthesis of 3-epi-1.alpha.,
25-dihydroxy-16-ene-cholecalciferol
[0258] Synthesis of 3-epi-1.alpha.,
25-dihydroxy-16-ene-cholecalciferol was carried out by reacting the
compound of formula II with the compound of formula IIIc followed
by removal of the protecting silyl groups with
tetra-n-butylammonium fluoride in tetrahydrofuram at room
temperature as depicted in the formula scheme below. The compound
of formula IIIc is disclosed in U.S. Pat. No. 5,145,846 (Sep. 8,
1992). 18
[0259] The synthesis was performed as follows. To a stirred, cold
(-78.degree. C.) solution of 582.9 mg (1.0 mmol) of the reagent
[3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-bis[[(1,1-dimethyl
dimethylsilyl]oxy]-2-methylenecy-clohexylidene]ethyl]diphenylphosphine
oxide (Ro 27-5110) in 5.0 mL of anhydrous THF was added 0.63 mL
(1.0 mmol) of a 1.6 M solution of n-butyllithium in hexanes. The
resultant deep red solution was stirred at -78.degree. C. for 20
minutes and treated with 175.3 mg (0.5 mmol) of [3aR-[1(R*),
3a.alpha.,
7a.beta.]]-1-[1,5-dimethyl-5-[(trimethylsilyl)oxy]hexyl]-3,3a,5,6,7,7a-he-
xahydro-7a-methyl-4H-inden-4-one in 2.0 mL of anhydrous THF. The
mixture was stirred at -78.degree. C. for 3.0 hours, allowed to
warm to room temperature and quenched with 10 mL of a 1:1 mixture
of 1.0 M Rochelle salt solution and 1.0 N KHCO.sub.3 solution.
After 15 minutes, the mixture was poured into 60 mL of ethyl
acetate and 40 mL of a 1:1 mixture of 1.0 M Rochelle salt solution
and 1.0 N KHCO.sub.3 solution. The organic phase was separated and
the aqueous phase was re-extracted with 3.times.50 mL of ethyl
acetate. The combined organic extracts were washed with 10% brine,
dried (Na.sub.2SO.sub.4), and evaporated to give 719 mg of a gum,
which was purified by flash chromatography on 50 grams of silica
gel (40-65 .mu.m mesh; 3.5 cm diameter column) with 5% ethyl
acetate in hexanes as eluent, taking 15-mL fractions. Fractions 3-5
were combined and evaporated to give 323 mg of a colorless gum. The
latter in 7.0 mL of THF was treated with 3.5 mL (3.5 mmol) of a 1.0
M solution of tetra-n-butylammonium fluoride in THF and the
solution was stirred at room temperature for 18 hours. It was
diluted with 15 mL of water, stirred for 15 minutes and poured into
a mixture of 75 mL of ethyl acetate and 60 mL of 10% brine. The
organic phase was separated and the aqueous phase was re-extracted
with 3.times.60 mL of ethyl acetate. The combined organic extracts
were washed with 4.times.75 mL of water, dried (Na.sub.2SO.sub.4)
and evaporated to give 191 mg of a gum, which was purified by flash
chromatography on 40 grams of silica gel (40-65 .mu.m mesh; 3.5 cm
diameter column) with ethyl acetate as eluent, taking mL fractions.
Fractions 10-25 were combined and evaporated. The residue was
dissolved in 10 mL of methyl formate, and the solution was filtered
through a 0.4 .mu.m filter and evaporated to give 164 mg of the
title compound as an amorphous solid: [(.alpha.)S(25,D)
-47.72.degree. (MeOH, c=1.01); UV (MeOH) .lambda..sub.max 263
(.epsilon.=17,027), 218 (12,368, shoulder) 209 (16, 082) nm; IR
(CHCl.sub.3) 3606, 3513 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta.
0.68 (3 H, s), 1.02 (6 H, d, J=6.8 Hz), 1.19 (6 H, s), 1.29-2.5 (H,
m), 2.57 (1 H, d, J=12.8), 2.84 (1 H, d, J=12.8), 4.05 (1 H, s),
4.31 (1 H, br t), 5.02 (1 H, s) 5.29 (1 H, s), 5.31 (1 H, s), 6.11
(1 H, d, J=11 Hz), 6.43 (1H, d, J=11 Hz); MS (EI) m/z 414.3
(M.sup.+, 60).
Example XXI
[0260] Synthesis of
3-epi-1,25-dihydroxy-16-ene-20-epicholecalciferol
[0261] Synthesis of 3-epi-1.alpha., 25-dihydroxy-16-ene-20-epi
cholecalciferol was carried out by reacting the compound of formula
II with the compound of formula IIId followed by removal of the
protecting silyl groups with tetra-n-butylammonium fluoride in
tetrahydrofuram at room temperature as depicted in the formula
scheme below. The compound of formula IIId is disclosed in EP
808,833. 19
[0262] The synthesis was performed as follows. To a stirred, cold
(-78.degree. C.) solution of 582.9 mg (1.0 mmol) of the reagent
[3S-(1Z,3a.alpha.,5a.alpha.)]-2-[3,5-bis
[[1,1-dimethyl)-dimethylsilyl]ox-
y]-2-methylenecyclohexyl-idene]ethyl]ethyl]diphenylphosphine oxide
in 5.0 mL of anhydrous THF was added 0.63 mL (1.0 mmol) of a 1.6 M
solution of n-butyllithium in hexanes and the resultant deep red
solution was stirred at -78.degree. C. for 15 mins. A solution of
175 mg (0.5 mmol) of [3aR-[1(S*), 3a.alpha.,
7a.beta.]]-1-[1,5-dimethyl-5-[(trimethylsilyl)oxy-
]hexyl]-3,3a,5,6,7,7a-hexahydro-7a-methyl-4H-inden-4-one in 2.5 mL
of anhydrous THF was added and the mixture was stirred at
-78.degree. C. for 2.5 hours and at room temperature for 30
minutes. It was quenched with 10 mL of a 1:1 mixture of 2.0 M
Rochelle salt solution and 2.0 N KHCO.sub.3 solution. After 15
minutes the mixture was poured into 60 mL of ethyl acetate and 40
mL of a 1:1 mixture of 2.0 M Rochelle salt solution and 2.0 N
KHCO.sub.3 solution. The organic phase was separated and the
aqueous phase was reextracted with 3.times.60 mL of ethyl acetate.
The combined organic extracts were washed with 100 mL of 10% brine,
dried (Na.sub.2SO.sub.4) and evaporated to give 702 mg of a gum,
which was purified by flash chromatography on silica gel (40-65
.mu.m mesh; 3.5 cm diameter column) with 7.5% ethyl acetate in
hexanes as eluent, taking 15-mL fractions. Fractions 4-7 were
combined and evaporated to give 310 mg of a colorless gum. The
latter was dissolved in 4.0 mL of THF, treated with 4.0 mL (4.0
mmol) of a 1.0 M solution of tetra-n-butylammonium fluoride in THF,
and the mixture was stirred at room temperature for 17 hours. The
mixture was diluted with 15 mL of water, stirred for 30 minutes and
poured into a mixture of 70 mL of ethyl acetate and 50 mL of 10%
brine. The organic phase was separated and the aqueous phase was
re-extracted with 3.times.60 mL of ethyl acetate. The combined
organic extracts were washed with 4.times.100 mL of water, dried
(Na.sub.2SO.sub.4) and evaporated to give 214 mg of a gum, which
was purified by flash chromatography on 45 g of silica gel (40-65
.mu.m mesh; 3.5 cm diameter column) with ethyl acetate as eluent,
taking 15-mL fractions. Fractions 13-26 were combined and
evaporated, and the residue was dissolved in 10 mL of methyl
formate. The solution was filtered through a 0.4 .mu.m filter and
the filtrate was evaporated to give 169 mg of the title compound as
a colorless foam: [.alpha.]S(25,D)-11.34.degree. (MeOH, c=0.67); UV
(MeOH) .lambda.max 264 (.epsilon.=17310), 218 (.epsilon.=13,161,
shoulder), 210 (.epsilon.=15,961) nm; IR (CHCl.sub.3) 3608 cm-1;
.sup.1H NMR .delta. 0.70 (3 H, s), 1.05 (3 H, d, J=6.8 Hz), 1.21 (6
H, s), 1.22-1.50 (13 H, m), 1.57-1.74(4 H, m), 1.97-2.23 (6 H, m),
2.35 (1 H, m), 2.45 (1 H, m), 2.55 (1 H, m), 2.64(1 H, d, J=6.8
Hz), 2.83 (1 H, d, J=12 Hz), 4.04(1 H, br s), 4.31 (1 H, br s),
5.02 (1 H, s), 5.31 (2 H, s), 6.11 (1 H, d, J=11 Hz), 6.43 (1 H, d,
J=11 Hz); MS (FAB) m/z 414 (M.sup.+, 12).
Example XXII
[0263] Synthesis of
3-epi-1,25-dihydroxy-16,(E)23-diene-cholecalciferol
[0264] Synthesis of
3-epi-1,25-dihydroxy-16,(E)23-diene-cholecalciferol was carried out
by reacting the compound of formula II with the compound of formula
IIIe followed by removal of the protecting silyl groups with
tetra-n-butylammonium fluoride in tetrahydrofuram at room
temperature as depicted in the formula scheme below. The compound
of formula IIIe is disclosed in U.S. Pat. No. 5,428,029 (Jun. 27,
1995) and U.S. Pat. No. 5,145,846 (Sep. 8, 1992). 20
[0265] The synthesis was performed as follows. To a stirred, cold
(-78.degree. C.) solution of 582.91 mg (1.0 mmol) of the reagent
[3S-(1Z,3a,5a)]-2-[3,5-bis[[(1,1-dimethylethyl)-dimethylsilyl]oxy]-2-meth-
ylenecyclo-hexylidene]ethyl]diphenylphosphine oxide (Ro 27-5110) in
6.0 mL of anhydrous THF was added 0.63 mL (1.00 mmol) of a 1.6 M
solution of n-butyllithium in hexanes and the resultant deep red
solution was stirred at -78.degree. C. for 20 minutes and treated
with 200 mg (0.57 mmol) of
1-[1,5-dimethyl-5-[(trimethylsilyl)oxy]-3-(E)hexenyl]-3,3a,5,6,7,7a-hexah-
ydro-7a-methyl-[3aR-[1(R*), 3a.alpha., 7a.beta.]]-4H-inden-4-one in
2.0 mL of anhydrous THF. The mixture was stirred at -78.degree. C.
for 3.0 hours, allowed to warm to room temperature and quenched
with 10.0 mL of a 1:1 mixture of 1.0 M Rochelle salt solution and
1.0 N KHCO.sub.3 solution. After 10 minutes, the mixture was poured
into 70 mL of ethyl acetate and 45 mL of a 1:1 mixture of 1.0 M
Rochelle salt solution and 1 N KHCO.sub.3 solution. The organic
phase was separated and the aqueous phase was reextracted with
3.times.60 mL of ethyl acetate, washed with 150 mL of 10% brine,
dried (Na.sub.2SO.sub.4) and evaporated to give a colorless gum,
which was purified by flash chromatography on 50 g of silica gel
(40-60 m.mu. mesh; 3.5 cm diameter column) with 7.5% ethyl acetate
in hexanes as eluent, taking 15-mL fractions. Fractions 5-10 were
combined as evaporated to give 382 mg of a colorless gum. The
latter was dissolved in 6.0 mL of THF, treated with 5.0 mL (5.0
mmol) of a 1.0 M solution of tetra-n-butylammonium fluoride in THF,
and the solution was stirred at room temperature for 17 hours. It
was diluted with 10 mL of water and poured into a mixture of 75 mL
of ethyl acetate and 50 mL of 10% brine. The organic phase was
separated and the aqueous phase was reextracted with 3.times.75 mL
of ethyl acetate. The combined extracts were washed with
4.times.150 mL of water, dried (Na.sub.2SO.sub.4), and evaporated
to give 280 mg of a semi-solid, which was purified by flash
chromatography on 50 g of silica gel (40-60 .mu.m mesh; 3.5 cm
diameter column) with 1.0% 2-propanol in ethyl acetate as eluent,
taking 15-mL fractions. Fractions 17-29 were combined and
evaporated to give 204 mg of a solid, which was dissolved in 10 mL
of anhydrous methyl formate. The solution was filtered through a
0.45 .mu.m filter and the filtrate was concentrated to ca. 5.0 mL
and then diluted with 0.5 mL of hexane. The solution was left at
0.degree. C. overnight and the crystals were collected by
filtration and dried under high vacuum to give 147 mg of the title
compound, mp 106-109.degree. C.; [.alpha.]S(25,D) -64.24 (MeOH,
c=0.33); UV (MeOH) .lambda..sub.max 263 (.epsilon.=18,445), 220
(shl, .epsilon.=12,524), 211 (shl, .epsilon.=17,145) nm; IR
(CHCl.sub.3) 3609 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.67
(3 H, s), 1.01 (3 H, d, J=6.8 Hz), 1.29 (6 H, s), 1.33 (1H, s, OH),
1.52 (1 H, m), 1.65-1.90 (4 H, m), 2.05-2.3 H, m), 2.37 (1 H, m),
2.45 (1 H, m), 2.64 (1 H, br d, OH), 2.83 (1 H, br d), 4.06 (1 H,
br s), 4.31 (1 H, br s), 5.02 (1 H, s), 5.31 (1 H, s), 5.59 (2 H,
m), 6.10 (1 H, d, J=11 Hz), 6.43 (1 H, d, J=11 Hz); MS
(electrospray) m/z 412 (M.sup.+).
Example XXIII
[0266] Synthesis of
3-epi-1,25-dihydroxy-16-ene-25-yne-cholecalciferol
[0267] Synthesis of
3-epi-1,25-dihydroxy-16-ene-25-yne-cholecalciferol was carried out
by reacting the compound of formula II with the compound of formula
IIIf followed by removal of the protecting silyl groups with
tetra-n-butylammonium fluoride in tetrahydrofuram at room
temperature as depicted in the formula scheme below. The compound
of formula IIIf is disclosed in U.S. Pat. No. 5,145,846 (Sep. 8,
1992) and U.S. Pat. No. 5,512,554 (Apr. 30, 1996). 21
[0268] The synthesis was performed as follows. To a stirred, cold
(-78.degree. C.) solution of 1.75 g (3.0 mmol) of the reagent
[3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-bis[[(1,1-dimethyl)-dimethylsilyl]oxy]-
-2-methylenecyclohex-ylidene]ethyl]diphenylphosphine oxide in 6.0
mL of anhydrous THF was added 1.9 mL (3.04 mmol) of a 1.6 M
solution of n-butyllithium in hexanes. The resultant red solution
was stirred at -78.degree. C. for 6 minutes and treated with 520 mg
(1.50 mmol) of [3aR-[1(R*), 3a.alpha.,
7a.beta.]]-1-[1,5-dimethyl-5-[(trimethylsilyl)oxy-
]-3-hexynyl]-3a,5,6,7,7a-hexahydro-7a-methyl-4H-inden-4-one in 3.0
mL of anhydrous THF. The mixture was stirred at -78.degree. C. for
2.5 hours, allowed to warm to 0.degree. C. and quenched with 10 mL
of a 1:1 mixture of 2.0 M Rochelle salt solution and 2.0 M solution
of KHCO.sub.3. After 10 minutes, the mixture was poured into 70 mL
of ethyl acetate and 50 mL of a 1:1 mixture of 2.0 M Rochelle salt
solution and 2.0 N KHCO.sub.3 solution. The organic phase was
separated and the aqueous phase was re-extracted with 3.times.60 mL
of ethyl acetate. The combined organic extracts were dried
(Na.sub.2SO.sub.4) and evaporated to give 1.35 of a gum, which was
purified by flash chromatography on 50 grams of silica gel (40-65
.mu.m mesh; 3.5 cm diameter column) with 8% ethyl acetate in
hexanes as eluent, taking 15-mL fractions. Fractions 4 and 5 were
combined and evaporated to give 153 mg of a colorless gum. The
latter was dissolved in 3.0 mL of THF, treated with 2.0 mL of a 1.0
M solution of tetra-n-butylammonium fluoride in THF and the
solution was stirred at room temperature for 17.0 hours. It was
diluted with 6.0 mL of water and 15 mL of ethyl acetate and stirred
for 15 minutes. The mixture was poured into 50 mL of ethyl acetate
and 50 mL of 10% brine. The organic phase was separated and the
aqueous phase was extracted with 3.times.60 mL of ethyl acetate.
The combined organic extracts were washed with 4.times.100 mL of
water, dried (Na.sub.2SO.sub.4) and evaporated to give 108 mg of a
gum, which was chromatographed on 40 grams of flash silica gel
(40-65 .mu.m mesh; 3.5 cm diameter column) with ethyl acetate as
eluent, taking 15-mL fractions. Fractions 12-18 were combined and
evaporated. The residue was dissolved in 10 mL of anhydrous methyl
formate and the solution was filtered through a 0.4 .mu.m filter.
Evaporation of the filtrate gave 84 mg of the title compound as a
colorless foam: [.alpha.]S(25,D) -47.80.degree. (MeOH, c=0.41); UV
(MeOH) 262 (68 =17,060), 210 (.epsilon.=12,094 shoulder), nm; IR
(CHCl.sub.3) 3603, 3516, 2224 cm.sup.-1; .sup.1HNMR (CDCl.sub.3)
.delta. 0.71 (3 H, s), 1.12 (3 H, d, J=6.8 Hz), 1.48 (6 H, s),
1.60-1.80 (5 H, m), 2.0 (3 H, m), 2.17-2.25 (3 H, m), 2.30-2.40 (3
H, m), 2.44 (1 H, dd, J=13,5), 2.57 (1 H, d, J=13 Hz), 2.64 (1 H,
d, J=6.8 Hz, OH), 2.84 (1 H, d, J=12 Hz), 4.07 (1 H, br s), 5.01 (1
H, s), 5.31 (1 H, s), 5.37 (1 H, s), 6.10 (1 H, d, J=11 Hz), 6.43
(1 H, d, J=11 Hz); MS (FAB) m/z 410.5 (M.sup.+, 80).
Example XXIV
[0269] Synthesis of
3-epi-1,25-dihydroxy-16,23E-diene-26,27-hexafluorochol-
ecalciferol
[0270] Synthesis of
3-epi-1,25-dihydroxy-16,23E-diene-26,27-hexafluorochol- ecalciferol
was carried out by reacting the compound of formula II with the
compound of formula IIIg followed by removal of the protecting
silyl groups with tetra-n-butylammonium fluoride in tetrahydrofuram
at room temperature as depicted in the formula scheme below. 22
[0271] The synthesis was performed as follows. To a stirred, cold
(-78.degree. C.) solution of 385.0 mg (0.66 mmol) of the reagent
[3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-bis[[(1,1-dimethylethyl)-dimethyl]oxy]-
-2-methylenecyclohexylidene]diphenylphosphine oxide in 4.0 mL of
anhydrous THF was added 0.42 mL (0.67 mmol) of a 1.6 M solution of
n-butyllithium in hexanes and the resultant deep red solution was
stirred at -78.degree. C. for 20 minutes and treated with 128.1 mg
(0.33 mmol) of [3aR-[1(R*), 3a.alpha.,
7a.beta.]]-3a,5,6,7,7a-hexahydro-7a-methyl-1-[6,6,6-trifluoro--
1-methyl-5-(trifluoro-methyl)-5-[(trimethylsilyl)oxyl]-3-hexenyl]-4H-inden-
-4-one in 2.0 mL of anhydrous THF. The mixture was stirred at
-78.degree. C. for 3.0 hours, allowed to warm to room temperature
and quenched with 5.0 mL of a 1:1 mixture of 1.0 M Rochelle salt
solution and 1.0 N KHCO.sub.3 solution. After 10 minutes, the
mixture was poured into 60 mL of ethyl acetate and 35 mL of a 1:1
mixture of 1.0 M Rochelle salt solution and 1 N KHCO.sub.3
solution. The organic phase was separated and the aqueous phase was
reextracted with 3.times.40 mL of ethyl acetate. The combined
organic extracts were washed with 150 mL of 50% brine, dried
(Na.sub.2SO.sub.4), and evaporated to give gum, which was purified
by flash chromatography on 45 g of silica gel (40-60 m.mu. mesh;
3.5 cm diameter column) with 20% ethyl acetate in hexanes as
eluent, taking 15-mL fractions. Fractions 4-6 were combined as
evaporated to give 244 mg of a colorless gum. The latter was
dissolved in 3.5 mL of THF, treated with 2.5 mL (2.5 mmol) of a 1.0
M solution of tetra-n-buthylammonium fluoride in THF, and the
solution was stirred at room temperature for 17 hours. It was
diluted with 5 mL of water, stirred for 15 minutes, and poured into
a mixture of 50 mL of ethyl acetate and 40 mL of 10% brine. The
organic phase was separated and the aqueous phase was reextracted
with 3.times.40 mL of ethyl acetate. The combined extracts were
washed with 4.times.100 mL of water, dried (Na.sub.2SO.sub.4), and
evaporated to give 176 mg of a gum, which was purified by flash
chromatography on 40 g of silica gel (40-60 m.mu. mesh; 3.5 cm
diameter column) with ethyl acetate as eluent, taking 15-mL
fractions. Fractions 8-15 were combined and evaporated and the
residue was dissolved in 10 mL of anhydrous methyl formate. The
solution was filtered through a 0.4 .linevert split.m filter and
the filtrate was evaporated to give 131 mg of the title compound as
a colorless solid: [.alpha.]S(25,D) -33.4.degree. (MeOH, c=0.53);
UV (MeOH) .lambda..sub.max 262 (.epsilon.=14,835), 218
(.epsilon.=10,960, shoulder), 209 (.epsilon.=13,793) nm; IR
(CHCl.sub.3) 3596, 2261 cm.sup.-1; .sup.1H NMR CDCl.sub.3) .delta.
0.67 (3 H, s), 1.04 (3 H, d, J=6.8 Hz), 1.50 (1 H, m), 1.6-1.85 (4
H, m) 2.05 (3 H, m), 2.25 (4 H, m), 2.40 (3 H, m), 2.55 (1 H, m),
2.74 (1 H, d, J=6.8 Hz) 2.84 (1 H, br d, J=13 Hz), 3.26 (1 H, s,
OH), 4.06 (1 H, br s), 4.33 (1 H, br s), 5.01 (H, s), 5.30 (1 H,
s), 5.32 (1 H, s), 5.60 (1 H, d, J=16 Hz), 6.10 (1 H, d, J=11 Hz),
6.16 (1 H, dt, J=16, 7.6) 6.43 (1 H, d, J=Hz); MS (FAB) m/z 520
(M.sup.+, 20).
Example XXV
[0272] Synthesis of
3-epi-1,25-dihydroxy-16-ene-23-yne-hexafluorocholecalc- iferol
[0273] Synthesis of
3-epi-1,25-Dihydroxy-16-ene-23-yne-hexafluorocholecalc- iferol was
carried out by reacting the compound of formula II with the
compound of formula IIIh as depicted in the formula scheme below.
23
[0274] The synthesis was performed as follows. To a stirred, cold
(-78.degree. C.) solution of 582.9 mg (1.0 mmol) of the reagent
[3S-(1Z,3.alpha.,5.alpha.)]-2-[3,5-bis[[(1,1-dimethylethyl)-dimethylsilyl-
]oxy]-2-methylenecyclohexylidene]ethyl]diphenylphosphine oxide in
5.0 mL of anhydrous THF was added 0.63 mL of a (1.0 mmol) 1.6 M
solution of n-butyllithium in hexanes and the resultant deep red
solution was stirred at -78.degree. C. for 17 minutes and treated
with 191.17 mg (0.5 mmol) of [3aR-[1(R*), 3a.alpha., 7a.beta.]]-3a,
5,6,7,7a-hexahydro-7a-methyl-1-[6,-
6,6-trifluoro-5-hydroxy-1-methyl-5-(trifluoromethyl)-3-hexynyl]-4H-inden-4-
-one in 2.5 mL of anhydrous THF. The mixture was stirred at
-78.degree. C. for 3.0 hours, allowed to warm to room temperature
and quenched with 10 mL of a 1:1 mixture of 2.0 M Rochelle salt
solution and 2.0 N KHCO.sub.3 solution. After 25 minutes, the
mixture was poured into a 1:1 mixture of 2.0 M Rochelle salt
solution and 2 N KHCO.sub.3 solution. The organic phase was
separated and the aqueous phase was re-extracted with 3.times.60 mL
of ethyl acetate. The combined organic extracts were washed with
50% brine, dried (Na.sub.2SO.sub.4), and evaporated to give 764 mg
of a gum, which was purified by flash chromatography on 50 g of
silica gel (40-60 m.linevert split. mesh; 3.5 cm diameter column)
with 7% ethyl acetate in hexanes as eluent, taking 15-mL fractions.
Fractions 5-12 were combined as evaporated to give 321 mg of a
colorless gum. The latter was dissolved in 4.0 mL of THF, treated
with 4.0 mL (4.0 mmol) of a 1.0 M solution of
tetra-n-buthylammonium fluoride in THF, and the solution was
stirred at room temperature for 18 hours. It was diluted with 10 mL
of water, stirred for 15 minutes, and poured into a mixture of 75
mL of ethyl acetate and 60 mL of 10% brine. The organic phase was
separated and the aqueous phase was reextracted with 3.times.60 mL
of ethyl acetate. The combined extracts were washed with
4.times.100 mL of water, dried (Na.sub.2SO.sub.4), and evaporated
to give 233 mg of a gum, which was purified by flash chromatography
on 40 g of silica gel (40-60 m.mu.; 3.5 cm diameter column) with
ethyl acetate as eluent, taking 15-mL fractions. Fractions 8-13
were combined and evaporated and the residue was dissolved in 10 mL
of anhydrous methyl formate. The solution was filtered through a
0.4 .mu.m filter and the filtrate was evaporated to give 181 mg of
the title compound as a colorless foam: [.alpha.]S(25,D)
-33.13.linevert split.(MeOH, c=0.51); UV (MeOH) .lambda..sub.max
263 (.epsilon.=17,431), 241 (.epsilon.=13,258, shoulder), 217
(.epsilon.=13,002, shoulder) 211 (.epsilon.=14,950) nm; IR
(CHCl.sub.3) 3594, 2261, 2239, 1195 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) d 0.70 (3 H,s), 1.14 (3 H, d, J=6.8 Hz), 1.50 (1 H,
m), 1.6-1.85 (4 H, m) 2.05 (3 H, m), 2.30 (2 H, m), 2.40 (5 H, m),
2.58 (1 H, m), 2.83 (2 H, m), 3.73 (1 H, s, OH) 4.07 (1 H, br s),
4.33 (1 H, br s), 5.01 (1 H, s), 5.30 (1 H, s), 5.41 (1 H, s), 6.10
(1 H, d, J=11 Hz), 6.43 (1 H, d, J=11 Hz); MS (EI) m/z 518.4
(M.sup.+, 60).
[0275] Equivalents
[0276] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *